Use of Anti-CD45 antibody drug conjugate (ADC) in cell therapy

Abstract

To provide a method for depleting CD45+ cells in a human patient undergoing chimeric antigen receptor (CAR) immunotherapy in order to promote acceptance of CAR-expressing immune cells.SOLUTION: Anti-CD45 antibody-drug conjugates (ADCs) are administered as a conditioning regimen to a human patient receiving autologous or allogeneic CAR-expressing immune cells, such that the CAR-expressing immune cells are accepted by the human patient. The compositions and methods of the present invention can be used in combination with CAR therapy to treat various pathological conditions, including autoimmune diseases and cancer.SELECTED DRAWING: None

Description

Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 789,462, filed on Jan. 7, 2019, and U.S. Provisional Application No. 62 / 845,829, filed on May 9, 2019. The content of each priority application is incorporated herein by reference.

Technical Field

[0002] The present invention generally relates to a method for promoting the engraftment of immune cells expressing a chimeric antigen receptor (CAR) in a human subject through the use of an anti-CD45 antibody-drug conjugate (ADC).

Background Art

[0003] Chimeric antigen receptor (CAR) therapy is an immunological treatment that uses lymphocytes derived from a patient or an allogeneic donor that have been engineered to destroy cells expressing a specific antigen associated with a particular disease such as cancer. For example, in cancer, CAR therapy marshals and enhances the power of the patient's immune system to attack the tumor. In recent years, this immunotherapy has received attention as a promising and revolutionary treatment method. CAR therapy is based on immune cells such as T cells expressing a CAR, and the CAR generally combines an extracellular antigen-binding domain such as an scFv with a cytoplasmic active signaling domain and a "co-stimulatory" domain that send signals into the cell from a surface receptor. Thus, when an immune cell such as a T cell expresses a CAR, the immune cell can recognize and kill cells expressing the antigen targeted by the antigen-binding domain of the CAR (e.g., a tumor-associated antigen) (Geyer and Bre ​ntjens (2016) Cytotherapy 18(11):1393-140 (page 9).

[0004] Although CAR therapy is a very powerful technique, it is associated with serious potential risks and adverse side effects ( Kay and Turtle (2017) Drugs 77(3):237-245 page; Hill et al. (2018) Blood 131:121-130 page). Lymphodepleting chemotherapy is commonly used as a conditioning treatment in combination with CAR therapy to minimize the rejection of CAR-expressing cells by the treated patients (Wei et al. (2017) Exp Hematol Oncol. 6 :10). For example, the combination of the lymphodepleting agents fludarabine and cyclophosphamide has improved the duration of CAR-T cells in recipient patients (Tur tle et al. (2016) J Clinic Invest 126(6): 2123; see also US20170368101). Conditioning therapy improves the efficacy of CAR- T cells, but lymphodepleting chemotherapy is often associated with serious negative side effects . SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] The present disclosure provides a conditioning regimen that can be used in conjunction with chimeric antigen receptor (CAR) therapy to facilitate the acceptance of CAR-expressing immune cells. The methods described herein can be used to facilitate the acceptance of either autologous CAR-expressing immune cells or allogeneic CAR-expressing immune cells. Conventionally, the acceptance of such cells has been achieved using lymphodepleting chemotherapy treatments for use. ​​has been achieved. This specification describes an improved method for promoting the acceptance of CAR-expressing cells in recipient patients. An improved method for promoting the acceptance of CAR-expressing cells in recipient patients is described.

[0006] In a first aspect, the present disclosure provides a method for promoting the acceptance of immune cells expressing a chimeric antigen receptor (CAR) in a human subject having cancer or an autoimmune disease, the method comprising: , (a) administering an anti-CD45 antibody-drug conjugate (ADC) to a human subject having cancer or an autoimmune disease, wherein the anti-CD45 ADC comprises an anti-CD45 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and (b) administering a therapeutically effective amount of immune cells expressing the CAR to the human subject, wherein the CAR comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain that binds to a tumor antigen expressed on the surface of cells or an antigen associated with an autoimmune disease expressed on the surface of cells; characterized by a method comprising. In one embodiment, the human subject is not administered alemtuzumab before, simultaneously with, or after step (b). In another embodiment, the human subject is not administered a lymphocyte-depleting chemotherapeutic agent before, simultaneously with, or after step (b). In yet another embodiment, the lymphocyte-depleting chemotherapeutic agent is fludarabine, cyclophosphamide, bendamustine, and / or pentostatin.

[0007] In certain embodiments, the method comprises administering the anti-CD45 ADC to the human subject before step (b).

[0008] In certain other embodiments, the method comprises administering the anti-CD45 ADC to the human subject about 12 hours to about 21 days (e.g., about 12 hours before, about 13 hours before, about 14 hours before, about 15 hours before, about 16 hours before, about 17 hours before, about 18 hours before, about 19 hours before, about 20 hours before, about 21 hours before, about 22 hours before, about 23 hours before, about 24 hours before, about 2 days before, about 3 days before, about 4 days before, about 5 days before, about 6 days before, about 7 days before, about 8 days before, about 9 days before, about 10 days before, about 11 days before, about 12 days before, about 13 days before, about 14 days before, about 15 days before, about 16 days before, about 17 days before, about 18 days before, about 19 days before, about 20 days before, or about 21 days before) prior to step (b).

[0009] In certain embodiments, the immune cells are allogeneic cells or autologous cells. In yet another embodiment, the allogeneic cells are allogeneic T cells or allogeneic NK cells.

[0010] In certain embodiments, the therapeutically effective amount of the allogeneic cells that express the CAR is about 1× 10 4 to about 1.0×10 8 cells / kg (e.g., about 1×10 4 to about 1×10 8 cells / kg 、about 1×10 4 to about 1×10 7 cells / kg、about 1×10 4 to about 1×10 6 cells / kg、about 1×10 4 to about 1×10 5 cells / kg、about 1×10 5 to about 1×10 8 cells / kg、about 1× 10 6 to about 1×10 8 cells / kg、or about 1×10 7 to about 1×10 8 cells / kg). ).

[0011] In another aspect, the present disclosure is a method of treating a patient having a tumor, the method comprising , (i) administering an anti-CD45 ADC, wherein the anti-CD45 ADC is an anti-CD45 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker ; and (ii) administering to the patient from about 1×10 to about 1×10 6 engineered CAR 8 T cells / kg (e.g., from about 1×10 to about 2×10 6 ; from about 2×10 6 to about 3×10 6 ; from about 6 3×10 to about 4×10 6 ; from about 4×10 6 to about 5×10 6 ; from about 5×10 6 to about 6×10 6 ; from about 6×10 6 to about 7×10 6 ; from about 7×10 6 to about 8×10 6 ; from about 8×10 6 to about 9×1 6 0 ; from about 9×10 6 to about 1×10 6 ; from about 1×10 7 to about 2×10 7 ; from about 2×10 7 to about 3 7 ×10 ; from about 3×10 7 to about 4×10 7 ; from about 4×10 7 to about 5×10 7 ; from about 5×10 7 to about 6×10 7 ; from about 6×10 to about 7×10 7 ; from about 7×10 7 to about 8×10 7 ; from about 8×10 7 to about 9×10 7 ; from about 9×1 0 7 to about 1×10 8 ; from about 1×106 , about 1×10 7 , or about 1×10 8 of engineered C AR T cells / kg); characterized by a method comprising administering a therapeutically effective amount. In one embodiment form, the therapeutically effective amount of the engineered CAR T cells is about 1×10 6 or about 2 ×10 6 cells / kg. In yet another embodiment, the anti-CD45 ADC is administered to the patient as a single dose or as multiple doses.

[0012] In certain embodiments, the anti-CD45 antibody or antigen-binding fragment thereof each has a heavy chain variable region comprising CDR1, CDR2 and CD R3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively. In another embodiment the anti-CD45 antibody or antigen-binding fragment thereof is chimeric or humanized . In other embodiments, the anti-CD45 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the anti-CD45 antibody or antigen-binding fragment thereof is of the IgG 1 isotype or the IgG4 isotype.

[0013] In certain embodiments, the cytotoxin is an anti-mitotic agent, a ribosome-inactivating protein (RIP) (e.g., Shiga toxin), or an RNA polymerase inhibitor. In other embodiments

[0014] (e.g., Shiga toxin), or an RNA polymerase inhibitor. In other embodiments In this case, the RNA polymerase inhibitor is amatoxin. In another embodiment, the R NA polymerase inhibitor is amanitin. In another embodiment, the amanitin is α -amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, proamanullin, and derivatives thereof. selected from the group consisting of:

[0015] In some embodiments of any of the above aspects, the cytotoxin is amatoxin , and the antibody or antigen-binding fragment thereof is conjugated to amatoxin via a linker and a chemical moiety to form an ADC represented by the formula Ab-Z-L-Am, where Ab is an antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is amatoxin. In some embodiments, the amatoxin is conjugated to the linker. In some embodiments, the amatoxin-linker -conjugate Am-L-Z is represented by formula (I):

Chemical formula

[0016] In other embodiments, the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). In yet another embodiment, the antimitotic agent is pyrrolobenzodiazepine (PBD) or calicheamicin.

[0017] ​ In certain embodiments, the linker of the ADC, together with the reactive substituent Z’, is N-beta-maleimidopropyl-Val-Ala-para-aminobenzyl (BMP-Va l-Ala-PAB).

[0018] In certain embodiments, the ADC has a serum half-life of 3 days or less.

[0019] In certain embodiments, the extracellular domain of the CAR comprises a scFv antibody or a single-chain T cell receptor (scTCR).

[0020] In certain embodiments, the extracellular domain comprises a non-immunoglobulin scaffold protein.

[0021] In certain embodiments, the tumor antigen is selected from the group consisting of CD19, CD22, CD30, CD7, BC MA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, C D38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI , CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123.

[0022] In certain embodiments, the cytoplasmic domain of the CAR comprises a CD28 cytoplasmic signaling domain, a CD3 zeta cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain, and / or a CD137 (4-1BB) cytoplasmic signaling domain.

[0023] In certain embodiments, the cytoplasmic domain of the CAR comprises a CD3 zeta cytoplasmic signaling domain.​​

[0024] In one embodiment, the anti-CD45 ADC is administered to the subject at a therapeutically effective amount prior to CAR therapy such that the hematopoietic stem cell (HSC) level is maintained in the patient while lymphocytes are depleted. In one embodiment, the level of HSCs in the subject is about 70% or more compared to the level of HSCs prior to anti-CD45 AD treatment in the subject . In one embodiment, the level of HSCs in the subject is about 80% or more compared to the level of HSCs prior to anti-CD45 AD treatment in the subject . In one embodiment, the level of HSCs in the subject is about 90% or more compared to the level of HSCs prior to anti-CD45 AD treatment in the subject . In one embodiment, the level of HSCs in the subject is about 90% or more compared to the level of HSCs prior to anti-CD45 AD treatment in the subject . In one embodiment, the level of HSCs in the subject is about 90% or more compared to the level of HSCs prior to anti-CD45 AD treatment in the subject .

[0025] In certain embodiments, the human subject having cancer is leukemia, advanced adult cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple negative breast cancer, hematopoietic / lymphoid cancer, liver metastasis of colon cancer, small cell lung cancer, non-small cell lung cancer, B-cell lymphoma, recurrent or refractory B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, recurrent or refractory diffuse large B-cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, recurrent mediastinal / refractory mediastinal large cell B-cell lymphoma (recurrent mediastinal, refractory mediastinal large B-cell lymphoma), large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, recurrent or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma mediastinal, refractory mediastinal large B-cell lymphoma), large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, recurrent or refractory mediastinal, refractory mediastinal large B-cell lymphoma), large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, recurrent or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma , refractory non-Hodgkin lymphoma, colorectal carcinoma, gastric cancer, pancreatic carcinoma, triple-negative invasive breast cancer, renal cell carcinoma, squamous cell lung cancer, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B -cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia , B-cell prelymphocytic leukemia, pediatric acute lymphoblastic leukemia, refractory pediatric acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prelymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed multiple myeloma, refractory multiple myeloma , multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone, malignant glioma of the brain, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasm, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumor, progressive solid tumor, mesothelin-positive tumor, hematologic malignancy, and other advanced malignancies.

[0026] In certain embodiments of any of the above aspects, the anti-CD45 antibody or antigen-binding fragment thereof comprises a combination of CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions) as set forth in Table 4 below. In certain embodiments, the anti-CD45 antibody or antigen-binding fragment thereof comprises a combination of a heavy-chain variable region and a light-chain variable region as set forth in Table 4 below.

[0027] In certain embodiments of any of the above aspects, the anti-CD45 ADC is administered to the subject in a therapeutically effective amount such that the hematopoietic stem cell ( HSC) level is maintained in the subject. In one embodiment, the level of HSC in the subject is the anti-CD45 in the subject It is about 70% or more compared to the level of HSCs before ADC treatment. In one embodiment, the level of HSCs in the subject is about 80% or more compared to the level of HSCs before anti-CD45 ADC treatment in the subject. In one embodiment, the level of HSCs in the subject is about 90% or more compared to the level of HSCs before anti-CD45 ADC treatment in the subject.

[0028] In certain embodiments of any of the above aspects, the anti-CD45 ADC treatment is administered in combination with T cell depletion therapy. In one embodiment, the T cell depletion therapy is administered before the administration of the anti-CD45 ADC treatment, simultaneously with the administration of the anti-CD45 ADC treatment, or after the administration of the anti-CD4 5 ADC treatment. In one embodiment, the T cell depletion therapy comprises an agent that binds to an antigen expressed on the cell surface of human T cells. In one embodiment, the T cell depletion therapy comprises an agent that binds to an antigen expressed on the cell surface of activated human T cells. In one embodiment, the T cell depletion therapy comprises an anti-CD4 antibody. In one embodiment, the T cell depletion therapy comprises an anti-CD8 antibody. In one embodiment, the T cell depletion therapy comprises an anti-CD137 antibody. In one embodiment, the T cell depletion therapy comprises an anti-CD52 antibody. In one embodiment, the T cell depletion therapy comprises an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD137 antibody, and / or

[0029] an anti-CD52 antibody. In one embodiment, the anti-CD52 antibody is alemtuzumab. In one embodiment, the ATG is rabbit ATG (rATG). In one embodiment, the AT​ G is a horse ATG (eATG).

[0030] In one embodiment, the T cell depletion therapy includes total body irradiation (TBI).

[0031] In one embodiment, the anti-CD45 ADC of lymphocyte depletion amount is administered.

[0032] In one embodiment, the human subject does not develop neutropenia after administration of the immune cells expressing the CAR. In certain embodiments, neutropenia is defined as a human subject having an absolute neutrophil count (ANC) of less than about 1500 (about 1500 / microliter) per microliter (e.g., less than about 1500 / μL, less than about 14 00 / μL, less than about 1300 / μL, less than about 1200 / μL, less than about 1100 / μL , less than about 1000 / μL, less than about 900 / μL, less than about 800 / μL, less than about 700 / μL , or less than about 600 / μL).

[0033] In one embodiment, the human subject does not develop severe neutropenia after administration of the immune cells expressing the CAR. In certain embodiments, the severe neutropenia is defined as an ANC of less than about 500 / μL (less than about 500 / μL, less than about 450 / μL, less than about 400 / μL, less than about 350 / μL, less than about 300 / μL, less than about 250 / μL, less than about 200 / μL, less than about 150 / μL, or less than about 100 / μL).

[0034] In one embodiment, the administration of the anti-CD45 ADC is effective to increase the level of one or more CAR-T engraftment cytokines in the human subject. In certain embodiments , the CAR-T engraftment cytokine is IL-15 or IL-7.

[0035] In one embodiment, the administration of the anti-CD45 ADC does not substantially increase the levels of one or more cytokines in the human patient with cytokine release syndrome (CRS). In certain embodiments, the administration of the anti-CD45 ADC does not substantially increase the levels of one or more cytokines in a human subject with cytokine release syndrome (CRS). In certain embodiments, the one or more CRS-cytokines are IFNγ, IL-10, IL-6, IL-8, MIP-1 α, MIP-1β, or IL-10. In certain embodiments, the one or more CRS-cytokines are IFNγ, IL-10, IL-6, IL-8, MIP-1 α, MIP-1β, or IL-10. In certain embodiments, the one or more CRS-cytokines are IFNγ, IL-10, IL-6, IL-8, MIP-1 BRIEF DESCRIPTION OF THE DRAWINGS

[0036]

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[0037] The present disclosure provides a method for treating a human subject undergoing CAR therapy by administering an anti-CD45 antibody-drug conjugate (ADC) to the patient undergoing CAR therapy, thereby reducing the level of chimeric antigen receptor A method for promoting the acceptance of immune cells (autologous or allogeneic) expressing a (CAR) is provided. The methods disclosed herein do not rely on (or alternatively reduce the use of) lymphodepleting chemotherapy, which is commonly used as a conditioning therapy to reduce the rejection of CAR-expressing immune cells, and can be used to improve the acceptance of autologous or allogeneic immune cells (e.g., T cells). (or alternatively reduce its use), and can be used to improve the acceptance of autologous or allogeneic immune cells (e.g., T cells ).

[0038] I. Definitions

[0039] As used herein, the term "about" refers to a value within 5% of the stated value.

[0040] As used in the context of transplantation, the term "allogeneic" is used to define cells (or tissues or organs) that are transplanted from a donor of the same species but a different individual to a recipient.

[0041] As used herein, the term "autologous" refers to cells or grafts in which the donor and recipient are the same individual.

[0042] As used herein, the term "xenogeneic" refers to cells in which the donor species and the recipient species are different.

[0043] As used herein, the term "immune cell" is intended to include, but is not limited to, cells of hematopoietic origin that play a role in the immune response. Immune cells include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. In one embodiment, natural killer cells are included. killer cells are included. Natural killer cells are well known in the art. In one embodiment, natural ​The killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. The immune cells can be allogeneic or autologous. In one embodiment, the immune cells are T cells .

[0044] The "engineered cell" means any cell of any organism that has been modified, transformed, or engineered by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. The isolated cells, host cells, and genetically engineered cells of the present disclosure include a DNA or RNA sequence encoding a CAR, and isolated immune cells such as NK cells and T cells that express the CAR on the cell surface. The isolated host cells and engineered cells can be used, for example, for enhancing NK cell activity or T lymphocyte activity, treating cancer, and treating autoimmune diseases. In one embodiment, the engineered cells include immune cells such as T cells or natural killer (NK cells). Cells expressing a chimeric antigen receptor (CAR) are examples of engineered cells.

[0045] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or immunologically reacts with a particular antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, hetero conjugate antibodies (e.g., bi-, tri- and quad-specific antibodies), diabodies, triabodies as long as they exhibit the desired antigen-binding activity, ​​​​​​ Di, as well as tetravalent antibodies), and antibody fragments (i.e., the antigen-binding fragments of antibodies) (e.g., Fab’, F(ab’)2, Fab, Fv, rlgG, and s cFv fragments).

[0046] The antibodies of the present disclosure are generally isolated or recombinant. As used herein when referring to a “isolated” polypeptide (e.g., an antibody), it means a polypeptide (e.g., an antibody) that has been identified, separated, and / or recovered from the cells or cell culture in which the polypeptide was expressed. Typically, an isolated antibody is prepared by at least one purification step. Thus, an “isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigen specificities. For example, an isolated antibody that specifically binds to CD45 is substantially free of antibodies that specifically bind to antigens other than CD45. Generally, an antibody comprises two heavy chains and two light chains that include an antigen-binding region. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3.

[0047] Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus towards the carboxyl terminus and are arranged Previously arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 The variable regions of the heavy and light chains each contain a binding domain that interacts with an antigen The constant region of the antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). can be mediated

[0048] As used herein, the term "complementary determining region" (CDR) refers to the hypervariable regions found in both the light chain and heavy chain variable domains of an antibody

[0049] The more highly conserved portions of the variable domains are called framework regions (FR). The amino acid positions that delineate the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art Some positions within the variable domain can be considered to be within the hypervariable region under certain criteria, but can be considered outside the hypervariable region under different criteria, and can be regarded as hybrid hypervariable positions One or more of these positions can also be considered part of the extended hypervariable region. The antibodies described herein can contain modifications at these hybrid hypervariable positions. The variable domains of native heavy and light chains each contain four framework regions that predominantly adopt a β-sheet structure and are connected by three CDRs, which form loops that connect the β-sheet structures and, in some cases, form part of the β-sheet structure. The CDRs in each chain are arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 by the framework regions and are connected to each other ​​​​​​held together in proximity and, together with CDRs from other antibody chains, form the target binding site of the antibody and contribute to (see Kabat et al., Sequences of Protein s of Immunological Interest, National Institutes of Health, Bethesda , Maryland, 1987). In certain embodiments, the immunoglobulin amino acid residue numbering is according to the Kabat et al. immunoglobulin amino acid residue numbering system, unless otherwise indicated (however, any antibody numbering scheme including, but not limited to, IMGT and Chothia can be utilized).

[0050] As used herein, the term “antigen-binding fragment” refers to one or more portions of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibody fragments include, for example, Fab, F (ab’)2, scFv, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to:( i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab’)2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody, (v) a dAb containing the VH and VL domains; (vi )V domains; (v) a dAb containing the VH and VL domains; (vi )V HdAb fragments consisting of domains (see, e.g., Ward et al., Na ture 341:544-546, 1989); (vii) dAbs consisting of VH or VL domains; (viii) isolated complementarity determining regions (CDRs); (ix) combinations of two or more (e.g., 2, 3, 4, 5, or 6) isolated CDRs optionally linked by synthetic linkers. Further, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be linked by a linker using recombinant methods that allows the VL and VH regions to pair to form a single protein chain that makes a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 2 42:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988 ). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art. In one embodiment, the antibody fragment contains an Fc region. As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, where each polypeptide chain contains a V on the same peptide chain and can be produced by methods known in the art. In one embodiment, the antibody fragment contains an Fc region.

[0051] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, where each polypeptide chain contains a V on the same peptide chain Hand V L Linkers that are too short (e.g., linkers composed of 5 amino acids) to allow intramolecular association of the domains link the V and V H and V L domains. With this configuration each domain pairs with a complementary domain on a separate polypeptide chain to form a homodimeric structure Thus, the term "tribody" refers to a trivalent antibody comprising three peptide chains each of which is a V within the same peptide chain H and and V L Linkers that are too short (e.g., linkers composed of 1 - 2 amino acids) to allow intramolecular association of the domains link one V domain and one V H domain. For folding into the native structure, peptides so configured are usually trimerized L such that the V and V H domains of adjacent peptide chains are spatially close to each other (see, e.g., Holliger et al., Proc. Natl. Aca L d. Sci. USA 90: 6444 - 48, 1993).

[0052] As used herein, the term "bispecific antibody" refers to, for example, a monoclonal antibody (e.g., a deimmunized or humanized antibody) that can bind to at least 2 different antigens, or two different epitopes that may be present on the same or different antigens For example one binding specificity can be directed to an epitope on a hematopoietic stem cell surface antigen such as CD45 and the other can be to a different cell surface antigen or another cell surface protein (especially a cell on receptors or receptor subunits involved in signal transduction pathways that enhance cell proliferation In some embodiments, the binding specificity is , can be directed to unique, non-overlapping epitopes on the same target antigen (i.e., Biparatopic antibody.

[0053] As used herein, an "intact" or "full length" antibody is one that is held together by disulfide bonds. Two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH). The heavy chain constant region is composed of CH1, CH2, and CH Each light chain is composed of three domains: a light chain variable region (LCVR) and a light chain variable region (LCVR). The light chain constant region consists of a single domain (abbreviated as VL) and a light chain constant region. The VH and VL regions are composed of superunits called complementarity-determining regions (CDRs). These can be further subdivided into regions of variability and more conserved regions called framework regions (FR). Each VH and VL consists of three CDRs and four FRs. From the amino terminus to the carboxyl terminus, FR1, CDR1, FR2, CDR2, FR3 The heavy and light chain variable regions are arranged in the order of CDR1, CDR2, and FR4. It contains an active binding domain.

[0054] Also included herein are "conservative sequence modifications" of the sequences set forth in the SEQ ID NOs. and amino acid sequence modifications, and the antibody or Nucleotide and amino acid sequence modifications are also provided that do not impair the binding of the antibody containing the amino acid sequence to the antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, nucleotide and amino acid additions and deletions. For example, the modifications can be introduced into the SEQ ID NOs described herein by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, predicted non-essential amino acid residues in an anti-CD45 antibody are preferably replaced with another amino acid residue of the same side chain family. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and ​​​​​​​​​​​​ See, e.g., Burks et al., Proc. Natl. Acad. Sci. USA 94: 412 - 417 (1997).

[0055] As used herein, the terms “anti - CD45 antibody” or “antibody that binds CD45” or “anti - CD45 ADC” or “ADC that binds CD45” refer to an antibody or ADC that specifically binds to human CD 45. CD45 is found on the cell surface of cells such as lymphocytes. The amino acid sequence of human CD45 with respect to an anti - CD45 antibody (or anti - CD45 ADC).

[0056] As used herein, the term “specifically binds” refers to the ability of an antibody (or ADC) to recognize and bind to a specific protein structure (epitope), rather than to proteins in general. When an antibody is specific for an epitope “A”, in a reaction containing the label “A” and the antibody, if a molecule containing epitope A (or free unlabeled A) is present, the amount of labeled A that binds to the antibody decreases. As an example, when an antibody is labeled, if it competes with the corresponding unlabeled antibody and dissociates from the target, that antibody “specifically binds” to the target. In one embodiment, the antibody has a dissociation constant of at least about 10 M or less, about 10 M or less, about 10 M or less, about 10 M or less, about 10 M or less, about 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less (less than means a number smaller than 10 -12 such as 10 -1 3 ​) means dissociation constant (K for the target D ) when having, the antibody binds specifically to the target (e.g., C D45). In one embodiment, as used herein, "specific binding to CD45" or the term "binds specifically to CD45" binds to CD45 and has a dissociation constant (K of 1.0 × 10 determined by surface plasmon resonance -7 M or less D ) having refers to an antibody. In one embodiment, K D is determined according to standard biolayer interferometry (BLI). However, it should be understood that the antibody may be able to specifically bind to two or more antigens with related sequences. For example, in one embodiment, the antibody can specifically bind to both human and non-human (e.g., mouse or non-human primate) orthologs of CD45. In some embodiments, the anti-CD45 antibody binds specifically to the extracellular domains of various isoforms of human CD45 (e.g., CD45RA (Uniprot accession number: P08575-8; sequence number 20), CD45RO (NCBI accession number: NP_563578.2;

[0057] sequence number 21), CD45RB (NCBI accession number: XP_00671153 7.1; sequence number 22), CD45RAB (NCBI accession number: XP_006 711535.1; sequence number 23), CD45RBC (NCBI accession number: X P_006711536.1; sequence number 24) and CD45RABC (NCBI accession number NP_002829.3; sequence number 25)). Thus, in certain embodiments, the antibodies herein can specifically bind to each extracellular domain of . Thus, in certain embodiments, the antibodies herein A pan-specific anti-CD45 antibody (i.e., an antibody that specifically binds to the extracellular regions of all six human CD45 isoforms). is provided.

[0058] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, prepared by any method available in the art or known. Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. Unless otherwise specified, the term "monoclonal antibody" (mAb) is meant to include both intact molecules and antibody fragments (e.g., including Fab and F(ab')2 fragments) that can specifically bind to a target protein. including any eukaryotic, prokaryotic, or phage clone, prepared by any method available in the art or known derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, prepared by any method available in the art or known. Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. Unless otherwise specified, the term "monoclonal antibody" (mAb) is meant to include both intact molecules and antibody fragments (e.g., including Fab and F(ab')2 fragments) that can specifically bind to a target protein. including hybridoma, recombinant, and phage display techniques, or combinations thereof, using a variety of techniques known in the art prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. Unless otherwise specified, the term "monoclonal antibody" (mAb) is meant to include both intact molecules and antibody fragments (e.g., including Fab and F(ab')2 fragments) that can specifically bind to a target protein. Unless otherwise specified, the term "monoclonal antibody" (mAb) is meant to include both intact molecules and antibody fragments (e.g., including Fab and F(ab')2 fragments) that can specifically bind to a target protein. that can specifically bind to a target protein (e.g., including Fab and F(ab')2 fragments). are included).

[0059] As used herein, the term "chimeric" refers to an antibody having a variable sequence derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985 , Science 229(4719):1202-7; Oi et al., 1 986, BioTechniques 4:214-221; Gillies e t al., 1985, J. Immunol. Methods 125:19 , Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent No. 5,807,715; U.S. Patent No. 4,816,567; and 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent No. 5,807,715; U.S. Patent No. 4,816,567; and 1985, J. Immunol. Methods 125:191-202; U.S. Patent No. 5,807,715; U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816,397. See also U.S. Patent No. 4,816,397. See also U.S. Patent No. 4,816,397.

[0060] As used herein, "drug-to-antibody ratio" or "DAR" refers to the ratio of the amount of a drug conjugated to an antibody. This refers to the average number of cytotoxins (e.g., amatoxins) administered to a given ADC. R ranges from about 1 to about 8, depending on the number of antibody linkage sites. Higher loads are possible. The anti-CD45 ADCs described herein may comprise 1, 2, 3, 4, 5, 6, 7, or 8 DA Has R.

[0061] "Humanized" forms of non-human (e.g., murine) antibodies are derived from non-human immunoglobulins. Generally, a humanized antibody is an immunoglobulin that contains at least one classic sequence. Typically, it contains substantially all of the two variable regions and all or substantially all of the CDR regions. All of the FR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions Humanized antibodies also contain a small amount of human immunoglobulin constant region. At least a portion (Fc), typically consisting of at least one of the human immunoglobulin consensus sequences Methods for humanizing antibodies are known in the art. For example, Rie chmann et al., 1988, Nature 332:323-7; U.S. Patent No. 5,530,101, U.S. Patent No. 5,585,089, U.S. Patent No. 5,6 93,761, U.S. Patent No. 5,693,762 and U.S. Patent No. 6,180,370 No. (Queen et al.); EP239400; PCT Publication WO91 / 09967 ;U.S. Patent No. 5,225,539;EP592106;EP519596;Padla n, in 1991, Mol. Immunol., 28: 489-498; Studnic ka et al., in 1994, Prot. Eng. 7: 805-814; Rogu ska et al., in 1994, Proc. Natl. Acad. Sci. 91: 9 69-973; see also U.S. Patent No. 5,565,332.

[0062] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide comprising at least an extracellular domain capable of specifically binding to an antigen, a transmembrane domain, and at least one intracellular signaling domain. Generally, a CAR is a genetically engineered receptor that redirects the cytotoxicity of immune effector cells to cells presenting a given antigen. A CAR is a molecule that combines the specificity for a desired antigen (e.g., a tumor antigen) by an antibody with a T cell receptor activation intracellular domain to generate a chimeric protein that exhibits specific cellular immune activity. In certain embodiments, a CAR comprises an extracellular domain (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain, and an intracellular (cytoplasmic) signaling domain. When the antigen-binding domain of the CAR binds to the target antigen on the surface of the target cell, the CAR clusters and provides an activation stimulus to the cell containing the CAR. The main feature of a CAR is to utilize the cell-specific targeting ability of monoclonal antibodies, soluble ligands, and cell-specific co-receptors to redirect the specificity of immune effector cells and induce the production of molecules that can mediate proliferation, cytokine production, phagocytosis, and cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner. It is to have the ability to emit. In various embodiments, the CAR is a human CD45 An extracellular binding domain that specifically binds to, a transmembrane domain, and one or more intracellular sig nal transduction domains.

[0063] As used herein, the term "CAR therapy" refers to the administration to a human subject of immune cells engineered to express a CAR for the treatment of a given disease (e.g., cancer or autoimmune disease). CAR therapy refers to the specific treatment of a patient with engineered immune cells and is not intended to include therapies (e.g., lymphodepleting chemotherapy) commonly used in conjunction with CAR cell treatment. In particular, when the term "cell" is used throughout, populations of cells are also included in the term unless otherwise specified. For example, CAR therapy requires the administration of a population of engineered cells.

[0064] As used herein, the term "combination" or "combination therapy" refers to the use of two (or more) therapies in a single human patient. The term is not intended to refer to a combination composition. For example, described herein is a combination therapy that includes administering an anti-CD45 ADC and CAR therapy.

[0065] The term "conditioning" refers to the preparation of a patient who requires CAR therapy for an appropriate state. Conditioning as used herein includes, but is not limited to, reducing the number of endogenous lymphocytes, removing cytokine sinks, increasing the serum levels of one or more homeostatic cytokines or pro-inflammatory factors prior to T cell therapy. administering, enhancing the effector function of T cells administered after conditioning administering, enhancing the activation and / or availability of antigen-presenting cells, or any combination thereof is included.

[0066] "Depletion" in the context of the effect of an anti-CD45 antibody or ADC on CD45-expressing cells refers to a decrease in the number of CD45-expressing cells or the elimination of CD45-expressing cells.

[0067] As used interchangeably herein, the phrases "therapeutically effective amount" or "therapeutically effective dose" refer to a therapeutic agent (e.g., an anti-CD45 ADC) that, upon single or multiple administration to a patient, provides a desired treatment and achieves a desired result, or is sufficient to affect an autoimmune disease or cancer. The "therapeutically effective amount" of a therapeutic agent can vary depending on the individual's disease state, age, sex, and weight [[ID=2D]]in order to elicit a desired response in an individual. The term "therapeutically effective amount" includes an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition of the invention can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). In one embodiment, the therapeutically effective amount of the anti-CD45 ADC is a lymphodepleting dose. When a therapeutic amount is indicated, the exact amount of the composition of the invention can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). In one embodiment, the therapeutically effective amount of the anti-CD45 ADC is a lymphodepleting dose. is a lymphodepleting dose.

[0068] As used herein, the phrase "lymphodepleting dose" refers to an amount of a therapeutic agent (e.g., an anti-CD45 antibody or anti-CD45 ADC) effective to deplete lymphocytes of a subject without substantially depleting the subject's hematopoietic stem cells ( HSCs).

[0069] As used herein, the term "half-life" refers to the time it takes for the plasma concentration of an antibody drug in a subject (e.g., a human subject) to decrease by half or 50%. This 50% decrease in serum concentration reflects the amount of drug circulating.

[0070] As used herein, the terms "Fc", "Fc region", "Fc domain", and "IgG Fc domain" refer to a portion of an immunoglobulin (e.g., an IgG molecule) that correlates with the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region includes the C-terminal half of the two heavy chains of the IgG molecule linked by disulfide bonds. The Fc region has no antigen-binding activity but includes a carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor) (see below). For example, the Fc domain includes the second constant domain CH2 (e.g., residues 231-340 of EU position of human IgG1) and the third constant domain CH3 (e.g., residues 341-447 of EU position of human IgG1). As used herein, the Fc region includes the "lower hinge region" (e.g., residues 233-239 of EU position of human IgG1).

[0071] Fc can refer to this region alone or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms are observed at many positions in the Fc domain including but not limited to EU positions 270, 272, 312, 315, 356, and 358, and thus there may be minor differences between the sequences presented in this application and those known in the art. Thus, "wild-type IgG Fc domain" or " ​​​​​​​​​​​​​​​The "WT IgG Fc domain" refers to any naturally occurring IgG Fc region (i.e., any allele). The sequences of the heavy chains of human IgG1, IgG2, IgG3, and IgG4 can be found in many sequence databases, such as the Uniprot database (www.uni prot.org), under accession numbers P01857 (IGHG1_ HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_ HUMAN), and P01861 (IGHG1_HUMAN), respectively.

[0072] As used herein, the terms "modified Fc region" or "variant Fc region" refer to an IgG Fc domain containing one or more amino acid substitutions, deletions, insertions, or modifications (alterations) introduced at any position within the Fc domain. In certain embodiments, the variant IgG Fc domain contains one or more amino acid substitutions and has a reduced or eliminated binding affinity for Fc gamma R and / or C1q as compared to any wild-type Fc domain that does not contain such one or more amino acid substitutions. Further, Fc binding interactions are essential for various effector functions and downstream signaling events, including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, in certain embodiments, an antibody (e.g., an antibody, fusion protein, or conjugate) comprising a variant Fc domain has reduced ADCC and / or CDC as compared to a corresponding antibody having the same amino acid sequence otherwise, except that it does not contain one or more amino acid substitutions, deletions, insertions, Altered binding affinity for at least one or more Fc ligands (e.g., Fc gamma R) can be shown.

[0073] The variant Fc domain is defined according to the amino acid modifications that make up the variant Fc domain. For all amino acid substitutions described herein with respect to the Fc region numbering always follows the EU index as in Kabat. Thus, for example, D 265C is an Fc variant in which aspartic acid (D) at EU position 265 of the parental Fc domain is replaced with cysteine (C). Similarly, for example, D265C / L234A / L235A defines a variant Fc having substitutions at EU positions 265 (D to C), 234 (L to A) and 235 (L to A) relative to the parental Fc domain. Variants can also be designated by the final amino acid composition at the mutated EU amino acid positions. For example, the L234A / L235A mutant may be referred to as "LALA". As a further example, the E233P.L234V.L235A.delG236 (deletion of 236 ) mutant may be referred to as "EPLVLAdelG". As yet another example the I253A.H310A.H435A mutant may be referred to as "IHH". Note that the order in which substitutions are provided is arbitrary.

[0074] As used herein, the term "Fc gamma receptor" or "Fc gamma R" refers to any member of the protein family that binds to the Fc region of an IgG antibody and is encoded by the Fc gamma R gene. In humans, this family includes, but is not limited to the isoforms Fc gamma RIa, Fc gamma RIb and Fc gamma Ric of the Fc gamma R family​ Fc gamma RI (CD64); isoforms Fc gamma RIIa (including allotypes H131 and R131), Fc gamma RIIb (including Fc gamma RIIb-1 and Fc gamma RIIb-2), and Fc gamma RIIc; Fc gamma RII (CD32); isoforms Fc gamma RIIIa (including allotypes V158 and F158), and Fc gamma RIIIb (including allotypes Fc gamma RIIIb-NA1 and Fc gamma R IIIb-NA2); Fc gamma RIII (CD16); and any undiscovered human Fc gamma R or Fc gamma R isoform or allotype. The Fc gamma R can be derived from any organism including, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse Fc gamma R includes, but is not limited to, Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD 16), and Fc gamma RIII-2 (CD16-2), and any undiscovered mouse Fc gamma R or Fc gamma R isoform or allotype.

[0075] As used herein, the term "effector function" refers to biochemical events resulting from the interaction between the Fc domain and the Fc receptor. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. As used herein, an "effector cell" is a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, Large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and gamma delta T cells are included and can be derived from any organism including, but not limited to, humans, mice, rats, rabbits, and monkeys. Obtainable.

[0076] As used herein, the terms "silent," "silenced," or "silencing" refer to an antibody having a modified Fc region where binding to the Fc gamma receptor (FcγR) is reduced compared to binding of the same antibody with an unmodified Fc region to the same FcγR (e.g., when measured by BLI, binding to the FcγR is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% reduced compared to binding of the same antibody with an unmodified Fc region to the same FcγR). In some embodiments, the Fc-silencing antibody has no detectable binding to the FcγR. Binding of an antibody with a modified Fc region to the FcγR can be measured using any of a variety of techniques known in the art, such as, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373, pages 52 - 60, 2008; or radioimmunoassay (RIA)), or surface plasmon resonance assays or other kinetic-based assays of other mechanisms (e.g., BIACORE analysis or Octet analysis (ForteBIO)), and other methods (indirect binding assays ). Techniques, for example, include, but are not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373, pages 52 - 60, 2008; or radioimmunoassay (RIA)), or surface plasmon resonance assays or other kinetic-based assays of other mechanisms (e.g., BIACORE analysis; or Octet analysis (ForteBIO)), and other methods (indirect binding assays or other kinetic-based assays of other mechanisms (e.g., BIACORE (商標) analysis or Octet (商標) analysis (ForteBIO)), and other methods (indirect binding assays , competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration), etc. It can be determined by. These and other methods utilize labels for one or more of the components to be tested and / or include, but are not limited to, chromogenic labels, fluorescent labels, luminescent labels, or isotope labels and may employ various detection methods. A detailed description of binding affinity and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), which focuses on the interaction between antibodies and immunogens. An example of a competitive binding assay is a radioimmunoassay that includes incubation of a labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen and detection of the antibody bound to the labeled antigen. The affinity and dissociation rate of the antibody of interest for a specific antigen can be determined from the data by Scatchard plot analysis. Competition with a secondary antibody can also be determined using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound in the presence of increasing amounts of unlabeled secondary antibody. When used herein, the term "identical antibody containing an unmodified Fc region" refers to an antibody that lacks the described amino acid substitutions (e.g., D265C, H435A, L234A, and / or L 235A) but otherwise has the same amino acid sequence as the Fc-modified antibody being compared.

[0077] As used herein, the term "identical antibody containing an unmodified Fc region" refers to an antibody that lacks the described amino acid substitutions (e.g., D265C, H435A, L234A, and / or L 235A) but otherwise has the same amino acid sequence as the Fc-modified antibody being compared. It has the same amino acid sequence as the Fc-modified antibody being compared, except that it lacks the described amino acid substitutions (e.g., D265C, H435A, L234A, and / or L 235A).

[0078] As used herein, the terms "subject" and "patient" refer to an organism, such as a human, undergoing treatment for a particular disease or condition described herein.

[0079] As used herein, the term "endogenous" refers to a substance that is naturally found in a particular organism, such as a human patient, including molecules, cells, tissues, or organs (e.g., CD45+ immune cells such as endogenous lymphocytes).

[0080] As used herein, the term "sample" refers to a specimen obtained from a subject, such as blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic fluid, chorionic villus sample, and cells.

[0081] As used herein, the phrase "substantially removed from the blood" refers to the point in time after administration of a therapeutic agent (such as an anti-CD45 antibody or an antigen-binding fragment thereof) to a patient, when the concentration of the therapeutic agent in a blood sample isolated from that patient cannot be detected by conventional methods (e.g., the therapeutic agent cannot be detected above the noise threshold of a device or assay used to detect the therapeutic agent). Various techniques known in the art, such as ELISA-based detection assays known in the art or described herein, can be used to detect antibodies, antibody fragments, and protein ligands. Additional assays that can be used to detect an antibody or antibody fragment include, among others, immunoprecipitation techniques and immunoblot assays known in the art.

[0082] As used herein, "treating" or "treatment" refers to an improvement in the outcome of a disease, such as prolonging survival, reducing the incidence rate, and / or reducing side effects that are by-products of alternative therapies; as will be readily understood in the art, complete eradication of the disease is preferred but not a requirement of the treatment act. Beneficial or desirable clinical outcomes include, but are not limited to, promoting the acceptance of CAR-expressing immune cells (allogeneic or autologous - either of which may cause an immune response in patients receiving CAR therapy). As long as the methods of the present disclosure are directed to prevention of a disorder, it is understood that the term "preventing" does not require that the disease state be completely blocked. Rather, as used herein, the term prevention refers to the ability of one of skill in the art to identify a population susceptible to a disorder such that administration of a compound of the present disclosure can occur prior to the onset of the disease. This term does not mean that the disease state is completely avoided.

[0083] As used herein, the term "vector" includes nucleic acid vectors such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. Expression vectors described herein may include a polynucleotide sequence and additional sequence elements used, for example, for expression of a protein and / or integration of these polynucleotide sequences into the genome of a mammalian cell. Specific vectors that can be used for expression of a CAR or an antibody include plasmids containing regulatory sequences such as promoters and enhancer regions that direct gene transcription. Other useful vectors for antibody or CAR expression either increase the translation rate of these genes or result from gene transcription It contains polynucleotide sequences that improve the stability or nuclear export of mRNA. These sequences elements can, for example, be used to direct efficient transcription of a gene carried on an expression vector and may include 5' and 3' untranslated regions as well as polyadenylation signal sites. The expression vectors described herein may also include a polynucleotide encoding a marker for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin (nourseothricin ).

[0084] As used herein, the term "antibody-drug conjugate" or "ADC" refers to an antibody conjugated to a cytotoxin. An ADC is formed by the chemical linkage of a reactive functional group of an antibody or an antigen-binding fragment thereof to a suitable reactive functional group of another molecule, such as a cytotoxin described herein. The conjugate may include, for example, a linker between two molecules that are bound to each other (e.g., between an antibody and a cytotoxin). Examples of linkers that can be used in the formation of conjugates include peptide-containing linkers, such as those containing naturally occurring amino acids or non-naturally occurring amino acids (such as D-amino acids). The linker can be prepared using various strategies described herein and known in the art . The linker can be cleaved, depending on the reactive components therein, for example, by enzymatic hydrolysis, photolysis , hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (e.g., Leriche et a l). The linker can be cleaved, depending on the reactive components therein, for example, by enzymatic hydrolysis, photolysis , hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (e.g., Leriche et a l., Bioorg.Med.Chem., 20:571-582, 2012 See).

[0085] As used herein, the term "microtubule binding agent" refers to a compound that acts by disrupting the microtubule network essential for cell functions during mitosis and interphase. Examples of microtubule binding agents include, but are not limited to, maytansine, maytansinoids, and their derivatives (such as those described herein or known in the art), vinca alkaloids (such as vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine), taxanes (such as docetaxel and paclitaxel), macrolides (such as discodermolide, cochicine, epothilone, and their derivatives (such as epothilone B or its derivatives), etc.).

[0086] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by Amanita phalloides or a derivative thereof, e.g., a variant or derivative thereof that can inhibit RNA polymerase II activity. Also included are synthetic amatoxins (see, e.g., U.S. Patent No. 9,676,702, which is incorporated herein by reference). Amatoxins useful in combination with the compositions and methods described herein include, but are not limited to, the amatoxins of formulas (III), (IIIa), (IIIb), and (III c) described herein (e.g., α-amanitin, β-amanitin, γ-amanitin, ε- Amanitin, amanin, amanin amide, amanullin, amanullinic acid, or proamanullin and derivatives thereof). As described herein, amatoxin can be conjugated to an antibody or an antigen-binding fragment thereof, for example, via a linker moiety (L), thereby forming an ADC. Such an ADC is represented by the formula Ab-Z-L-Am, where Ab is an antibody or an antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is amatoxin. In some embodiments, amatoxin is conjugated to the linker. In some embodiments, the amatoxin-linker conjugate Am-L-Z is represented by formula (I) or ( IA), (IB), (IV), (IVA), or (IVB). Exemplary methods of conjugating amatoxin and linkers useful in such processes are described below. Exemplary linker-containing amatoxins useful for conjugating to an antibody or antigen-binding fragment according to the compositions and methods are also described herein . The term "acyl" as used herein refers to -C(=O)R, where, as defined herein, R is hydrogen (``aldehyde''), alkyl (e.g., C1-C alkyl), alkenyl (e.g., C2-C .

[0087] alkyl), alkynyl (e.g., C2-C alkynyl), carbocyclic (e.g., C3-C7 carbocyclic), aryl ( 12 alkyl), alkenyl (e.g., C2-C alkenyl), alkynyl (e.g., C2-C 12 alkynyl), carbocyclic (e.g., C3-C7 carbocyclic), aryl ( 12 alkynyl), carbocyclic (e.g., C3-C7 carbocyclic), aryl (e.g., C6-C aryl), heteroaryl (e.g., 5-10 membered heteroaryl 20 aryl), heteroaryl (e.g., 5-10 membered heteroaryl ) or is a heterocyclyl (e.g., 5- to 10-membered heterocyclyl). Non-limiting Examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl. are mentioned.

[0088] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon having 1 to 12 carbon atoms. Representative C1-C 12 alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while branched C1-C 12 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-but yl, -isopentyl, and 2-methylbutyl. C1-C 12 alkyl groups may be unsubstituted or substituted.

[0089] As used herein, the term "alkenyl" refers to an unsaturated moiety having at least one site, i.e., a carbon-carbon, sp2 double bond, and containing a normal, secondary, or tertiary carbon atom, and refers to a C2-C 12 hydrocarbon. Examples include, but are not limited to, the following: ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isob tylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2- methyl-2-butenyl, -2,3-dimethyl-2-butenyl, etc. Alkenyl groups may be unsubstituted or substituted. are mentioned.

[0090] As used herein, "alkynyl" refers to an unsaturated moiety having at least one site, i.e., carbon - C having a carbon, sp triple bond, and containing a normal, secondary, or tertiary carbon atom 2-C 12 refers to a hydrocarbon. Examples include, but are not limited to, acetylene (ac etylenic) and propargyl. The alkynyl group may be unsubstituted or substituted.

[0091] As used herein, "aryl" refers to a C6-C 20 carbocyclic aromatic group. Examples of the aryl group include, but are not limited to, phenyl, naphthyl, and anthracenyl group. The aryl group may be unsubstituted or substituted.

[0092] As used herein, "arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom (typically a terminal or sp 3 carbon atom) is substituted by an aryl radical. Typical arylalkyl groups include, but are not limited to benzyl, 2-phenylethane-1-yl, 2-phenylethene-1-yl, naphth ylmethyl, 2-naphthylethane-1-yl, 2-naphthylethene-1-yl, naphthobenz dyl, 2-naphthophenylethane-1-yl, etc. The arylalkyl group contains 6 to 20 carbon atoms. For example, the alkyl portion of the arylalkyl group (e.g., alkyl anyl, alkenyl, or alkynyl group, etc.) has 1 to 6 carbon atoms, and the aryl portion has 5 to 14 carbon atoms. The alkaryl group may be unsubstituted or substituted. As used herein, "cycloalkyl" refers to a saturated carbocyclic radical, which is monocyclic

[0093] ​It may be monocyclic or bicyclic. The cycloalkyl group includes a ring having 3 to 7 carbon atoms as a monocyclic ring , or a ring having 7 to 12 carbon atoms as a bicyclic ring. Examples of the monocyclic cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclo heptyl, and cyclooctyl. The cycloalkyl group may be unsubstituted or substituted .

[0094] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical, which may be monocyclic or bicyclic. The cycloalkenyl group includes a ring having 3 to 6 carbon atoms as a monocyclic ring , or a ring having 7 to 12 carbon atoms as a bicyclic ring. Examples of the monocyclic cycloalkenyl group include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl (1-cyclohex -1-enyl), 1-cyclohex-2-enyl (1-cyclohex-2-eny l), and 1-cyclohex-3-enyl (1-cyclohex-3-enyl). The cycloalkenyl group may be unsubstituted or substituted.

[0095] As used herein, "heteroalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom (typically a terminal or sp 3 carbon atom) is substituted with a heteroaryl radical. Typical heteroarylalkyl groups include, but are not limited to , 2-benzimidazolylmethyl, 2-furylethyl, etc. The heteroarylalkyl group contains 6 to 20 carbon atoms. For example, heteroarylalkyl The alkyl portion of the ~yl group (such as an alkanoyl, alkenyl or alkynyl group) has 1 to 6 carbon atoms, and the heteroaryl portion has 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S. The heteroaryl portion of the heteroarylalkyl group is a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms) or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms, and 1 to 3 heteroatoms selected from N, O, P and S) (for example: bicyclo[4,5], [5,5], [5, 6], or [6,6] systems).

[0096] As used herein, "heteroaryl" and "heterocycloalkyl" each refer to an aromatic or non-aromatic ring system, where one or more ring atoms are heteroatoms, such as nitrogen, oxygen, and sulfur. A heteroaryl or heterocycloalkyl radical has 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S. Heteroaryl or heterocycloalkyl has a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms, and 1 to 3 heteroatoms selected from N, O, P and S) , or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms, and 1 to 3 heteroatoms selected from N, O, P and S) (for example: bicyclo[4,5], [5,5], [5,6], or [6,6] systems). Heteroaryl and heterocyclo alkyl may be unsubstituted or substituted.

[0097] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A. ; "Principles of Modern Heterocyclic Chem istry” (W.A. Benjamin, New York, 1968), especially Chapters 1, 3 , 4, 6, 7 and 9; “The Chemistry of Heterocycl ic Compounds, A series of Monographs” (Jo hn Wiley & Sons, New York, 1950 - present), especially Volumes 13, 1 4, 16, 19 and 28; and J. Am. Chem. Soc. (1960) 8 2: 5566.

[0098] Examples of heteroaryl groups include, for example, but not limited to, pyridyl, thia zolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pi razolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indri l, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl , pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H - indolyl, 1 H - indazolyl, purinyl, 4H - quinolidinyl, phthalazinyl, naphthyridinyl, qui noxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH - carbazolyl, ca rbazolyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furoxanyl, phenoxazinyl, isochromanyl, chro manil, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzo triazolyl, benzisoxazolyl, and isatinoyl.

[0099] Examples of heterocycloalkyl include, for example, but not limited to, dihydropi Ridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidini lu, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tet rahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahyd roisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl , quinuclidinyl, and morpholinyl.

[0100] By way of example and not limitation, carbon-bonded heteroaryl and heterocycloalkyl are bonded at the 2, 3, 4, 5, or 6 positions of pyridine, the 3, 4, 5, or 6 positions of pyridazine, the 2, 4, 5, or 6 positions of pyrimidine, the 2, 3, 5, or 6 positions of pyrazine, furan, tetrahydro furan, thiophene, thiophene, pyrrole, or tetrahydropyrrole at the 2, 3, 4 or 5 positions, oxazole, imidazole, or thiazole at the 2, 4, or 5 positions, isoxazole, pyrazole, or isothiazole at the 3, 4, or 5 positions, aziridi ne at the 2 or 3 positions, azetidine at the 2, 3, or 4 positions, quinoline at the 2, 3, 4, 5, 6 , 7, or 8 positions, or isoquinoline at the 1, 3, 4, 5, 6, 7, or 8 positions. Further typically, carbon-bonded heterocycles include 2-pyridyl, 3-pyridyl, 4 -pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5- pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidi nil, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyra dinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0101] ​​​By way of example and not limitation, nitrogen-linked heteroaryl and heterocycloalkyl are attached to the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or beta-carboline. Further typically, as nitrogen-linked heterocycles, 1-aziridyl, 1-azetidyl (1-azetedyl), 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl are included. aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or beta-carboline. By way of example and not limitation, nitrogen-linked heteroaryl and heterocycloalkyl are attached to the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or beta-carboline. By way of example and not limitation, nitrogen-linked heteroaryl and heterocycloalkyl are attached to the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or beta-carboline. aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or beta-carboline.

[0102] As used herein, and as applicable to any of the foregoing alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc., "substituted" means that one or more hydrogen atoms are each independently replaced with a substituent. Typical substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH2, -NHR, -N(R)2, -N (R) 3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, + (R) 3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C (=O)NH2, -C(=O)N(R)2, -SO3-, -SO3H, -S(=O)2R (=O)NH2, -C(=O)N(R)2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH2, -S(=O)2N(R)2, -S(= O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P(=O)(OR) O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P(=O)(OR) 2. -PO3, -PO3H2, -C(=O)X, -C(=S)R, -CO2H, -CO2 R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O )NH2, -C(=O)N(R)2, -C(=S)NH2, -C(=S)N(R)2, - C(=NH)NH2, and -C(=NR)N(R)2 are included; where each X is F , Cl, Br and I, independently selected for each case; and each R is C1-C 12 alkyl, C6-C 20 aryl, C3-C 14 heterocycloalkyl or heteroaryl, protecting groups and prodrug moieties, independently selected for each case . If a group is described as "optionally substituted", in any case that group can be independently substituted by one or more of the above substituents for each case. Substitution can involve adjacent substituents undergoing ring closure (e.g., ring closure of adjacent functional substituents) to form lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals, etc. formed by the ring closure, for example, to provide protecting groups . Situations can be included.

[0103] It should be understood that the naming rules for certain radicals can include either mono-radicals or diradicals depending on the situation. For example, if a substituent requires two attachment points to the rest of the molecule , that substituent is understood to be a diradical. For example , substituents identified as alkyl that require two attachment points include diradicals such as -CH2-, -CH 2CH2-, -CH2CH(CH3)CH2-, etc. Other radicals ​​The naming rules clearly indicate that the radical is a diradical such as "alkylene", "alkenylene", "arylene", "hetero cycloalkylene", etc.

[0104] "Isomer" or "isomeride" refers to compounds that have the same molecular formula but differ in the order of bonding of their atoms or the arrangement of those atoms in space. Isomers with different arrangements of atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereoisomers", and stereoisomers that are mirror images that cannot be superimposed on each other are called "enantiomers" or sometimes "optical isomers". A carbon atom bonded to four different substituents is called a "chiral center". "Chiral isomers" mean compounds that have at least one chiral center. Compounds with multiple chiral centers can exist as individual diastereomers or as a mixture of diastereomers called a "mixture of diastereomers". When there is one chiral center, the stereoisomers can be characterized by the absolute configuration (R or S) of that chiral center . The absolute configuration refers to the spatial arrangement of the substituents bonded to the chiral center. The substituents bonded to the chiral center under consideration are ranked according to the Cahn-Ingold-Prelog Sequence Rule

[0105] (Cahn et al., Angew.Chem.Inter.Edit.196 6, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J.Ch em. In the case where there is one chiral center, the stereoisomers can be characterized by the absolute configuration (R or S) of that chiral center . The absolute configuration refers to the spatial arrangement of the substituents bonded to the chiral center. The substituents bonded to the chiral center under consideration are ranked according to the Cahn-Ingold-Prelog Sequence Rule (Sequence Rule of Cahn, Ingold and Prelog) and are ranked (Cahn et al., Angew.Chem.Inter.Edit.196 6, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J.Ch em. 1966, 78, 413; Cahn and Ingold, J.Ch em.Soc. 1951 (London), 612; Cahn et al., E xperientia 1956, 12, 81; Cahn, J.Chem.Edu c. 1964, 41, 116). A mixture containing equal amounts of the individual enantiomeric forms of opposite chirality is called a "racemic mixture". A mixture containing equal amounts of the individual enantiomeric forms of opposite chirality is called a "racemic mixture".

[0106] The compounds disclosed in this specification and the claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers of each compound may exist. The description of the compounds in this specification and the claims includes, unless otherwise specified, all enantiomers, diastereomers, and mixtures thereof. The compounds disclosed in this specification and the claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers of each compound may exist. The description of the compounds in this specification and the claims includes, unless otherwise specified, all enantiomers, diastereomers, and mixtures thereof. In the description of the compounds in this specification and the claims, unless otherwise specified, it is intended to include all enantiomers, diastereomers, and mixtures thereof. In the description of the compounds in this specification and the claims, unless otherwise specified, it is intended to include all enantiomers, diastereomers, and mixtures thereof. Furthermore, the description of the compounds in this specification and the claims is intended to include, unless otherwise specified, both the individual enantiomers and racemic or other mixtures of enantiomers. Furthermore, the description of the compounds in this specification and the claims is intended to include, unless otherwise specified, both the individual enantiomers and racemic or other mixtures of enantiomers. If the structure of a compound is depicted as a specific enantiomer, the disclosure of the present application is understood not to be limited to that specific enantiomer. Thus, the enantiomers, optical isomers, and diastereomers of each structural formula of the present disclosure are contemplated herein. In this specification, the structural formula of a compound may, for convenience, represent a certain isomer, but the present disclosure includes all isomers such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and it is understood that all isomers do not necessarily have the same level of activity. If the structure of a compound is depicted as a specific enantiomer, the disclosure of the present application is understood not to be limited to that specific enantiomer. Thus, the enantiomers, optical isomers, and diastereomers of each structural formula of the present disclosure are contemplated herein. In this specification, the structural formula of a compound may, for convenience, represent a certain isomer, but the present disclosure includes all isomers such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and it is understood that all isomers do not necessarily have the same level of activity. Accordingly, the enantiomers, optical isomers, and diastereomers of each structural formula of the present disclosure are contemplated herein. In this specification, the structural formula of a compound may, for convenience, represent a certain isomer, but the present disclosure includes all isomers such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and it is understood that all isomers do not necessarily have the same level of activity. In this specification, the structural formula of a compound may, for convenience, represent a certain isomer, but the present disclosure includes all isomers such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and it is understood that all isomers do not necessarily have the same level of activity. In this specification, the structural formula of a compound may, for convenience, represent a certain isomer, but the present disclosure includes all isomers such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and it is understood that all isomers do not necessarily have the same level of activity. The compounds may exist in different tautomeric forms. The compounds according to the present disclosure include, unless otherwise specified, all tautomers. The compounds may exist in different tautomeric forms. The compounds according to the present disclosure include, unless otherwise specified, all tautomers. The compounds may exist in different tautomeric forms. The compounds according to the present disclosure include, unless otherwise specified, all tautomers. It means. When the structure of a compound is depicted as a specific tautomer, the disclosure of this application is understood not to be limited to that specific tautomer.

[0107] Compounds of any formula described herein include the compounds themselves, as well as their salts, and where applicable, their solvates. For example, salts can be formed between anions and positively charged groups (such as amino groups) on the compounds of the present disclosure. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronic acid, glutaric acid, malic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, tosylate, salicylate, lactate, naphthalenesulfonic acid, acetate (such as trifluoroacetate salt), and the like. The term "pharmaceutically acceptable anion" refers to an anion suitable for forming pharmaceutically acceptable salts. Similarly, salts can also be formed between cations on the compounds of the present disclosure and negatively charged groups (such as carboxylates). Suitable cations include sodium ions, potassium ions, magnesium ions, calcium ions, and ammonium cations such as tetramethylammonium ions. Examples of some suitable substituted ammonium ions are ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzyl amine, choline, meglumine, tromethamine, and those derived from amino acids such as lysine and arginine. Also, salts containing quaternary nitrogen atoms of the compounds of the present disclosure are included. ​ is formed.

[0108] Examples of suitable inorganic anions include, but are not limited to, those obtained from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, phosphoric acid, and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, crotonic acid, edetic acid, ethanesulfonic acid, fumaric acid, gluceptonic acid, glucuronic acid, glycocholic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic, isethionic acid. Lactic acid, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, valeric, etc. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose.

[0109] Furthermore, the compounds of the present disclosure, for example, salts of the compounds, can exist in either the hydrated or anhydrous form, or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc. "Solvate" refers to a solvate addition form containing a stoichiometric or non-stoichiometric amount of a solvent. ​​​ means a state. Among compounds, there are those that capture solvent molecules in a certain molar ratio in the state of a crystalline solid and have the property of forming a solvate. When the solvent is water, the solvate formed is a hydrate and when the solvent is alcohol, the solvate formed is an alcoholate. A hydrate is formed by the combination of one or more water molecules and one molecule of a substance, and water retains its molecular state as H2O Hydrates refer to, for example, monohydrates, dihydrates, trihydrates, etc.

[0110] Furthermore, for the compounds represented by the formulas disclosed in this specification or salts thereof, crystal polymorphs may exist. It should be noted that the scope of the present disclosure includes any crystal form, crystal form mixture, or their anhydrides or hydrates.

[0111] When a substituent is depicted as a diradical (i.e., having two bonding points to the rest of the molecule), it should be understood that, unless otherwise specified, the substituent can be bonded in any directional arrangement at any time.

[0112] In the following section, a method based on the administration of anti - CD45 ADC to a human patient is provided to promote the acceptance of immune cells expressing CAR in CAR therapy. [[ID=3*]]

[0113] II. Methods of Treatment with Anti - CD45 Antibody - Drug Conjugates (ADCs) and Chimeric Antigen Receptor (CAR) Cells

[0114] A challenge in chimeric antigen receptor (CAR) therapy is to determine the means by which engineered CAR - expressing cells (e.g., CAR - T cells) are accepted by a human recipient. The acceptance of such engineered immune cells can affect the effectiveness of treatment and can also be harmful to the patient ​​ It may cause no side effects.

[0115] Lymphocyte depletion chemotherapy is a conventional method of suppressing the recipient's immune system to improve acceptance, but generally has harmful side effects. Described herein is a method for promoting the acceptance of CAR-expressing immune cells in human patients undergoing CAR therapy. The method described herein specifically targets CD45+ cells, such as CD45+ lymphocytes (e.g., T cells), in human patients undergoing CAR therapy and removes the CD45+ cells. The method disclosed herein is more targeted than lymphocyte depletion chemotherapy and provides a means that can use either autologous cells or allogeneic cells. The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). of the method, lymphocyte depletion chemotherapy is a conventional method of suppressing the recipient's immune system to improve acceptance, but generally has harmful side effects. Described herein is a method for promoting the acceptance of CAR-expressing immune cells in human patients undergoing CAR therapy. The method described herein specifically targets CD45+ cells, such as CD45+ lymphocytes (e.g., T cells), in human patients undergoing CAR therapy and removes the CD45+ cells. The method disclosed herein is more targeted than lymphocyte depletion chemotherapy and provides a means that can use either autologous cells or allogeneic cells. The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). therapy in human patients undergoing CAR therapy. The method described herein specifically targets CD45+ cells, such as CD45+ lymphocytes (e.g., T cells), in human patients undergoing CAR therapy and removes the CD45+ cells. The method disclosed herein is more targeted than lymphocyte depletion chemotherapy and provides a means that can use either autologous cells or allogeneic cells. The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). The method described herein specifically targets CD45+ cells, such as CD45+ lymphocytes (e.g., T cells), in human patients undergoing CAR therapy and removes the CD45+ cells. The method disclosed herein is more targeted than lymphocyte depletion chemotherapy and provides a means that can use either autologous cells or allogeneic cells. The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). 45+ lymphocytes (e.g., T cells) and removes the CD45+ cells. The method disclosed herein is more targeted than lymphocyte depletion chemotherapy and provides a means that can use either autologous cells or allogeneic cells. The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). The method disclosed herein is more targeted than lymphocyte depletion chemotherapy and provides a means that can use either autologous cells or allogeneic cells. The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). The method disclosed herein is more targeted than lymphocyte depletion chemotherapy and provides a means that can use either autologous cells or allogeneic cells. The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). The advantage of the method disclosed herein is that the treatment depletes lymphocytes in patients who need it (i.e., patients who need CAR treatment), but does not substantially deplete HSCs. For example, the method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). The method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). The method disclosed herein may be able to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system). It may be possible to deplete lymphocytes in patients who need it without inducing myelosuppression (e.g., bone marrow suppression that requires HSC transplantation to restore the patient's hematopoietic system).

[0116] Described herein is a method of administering an anti-CD45 antibody-drug conjugate (ADC) to deplete a population of CD45-specific cells (e.g., lymphocytes) in a patient undergoing CAR therapy to promote the acceptance and efficacy of CAR-expressing immune cells. The selective depletion of these specific CD45-expressing cells of the immune system reduces the risk of rejection of CAR-expressing immune cells for treating autoimmune diseases or cancer while improving overall survival and recurrence-free patient survival. The selective depletion of these specific CD45-expressing cells of the immune system reduces the risk of rejection of CAR-expressing immune cells for treating autoimmune diseases or cancer while improving overall survival and recurrence-free patient survival. The selective depletion of these specific CD45-expressing cells of the immune system reduces the risk of rejection of CAR-expressing immune cells for treating autoimmune diseases or cancer while improving overall survival and recurrence-free patient survival. The selective depletion of these specific CD45-expressing cells of the immune system reduces the risk of rejection of CAR-expressing immune cells for treating autoimmune diseases or cancer while improving overall survival and recurrence-free patient survival. The selective depletion of these specific CD45-expressing cells of the immune system reduces the risk of rejection of CAR-expressing immune cells for treating autoimmune diseases or cancer while improving overall survival and recurrence-free patient survival.

[0117] The risk of rejection of CAR-expressing immune cells remains high even after the administration of CAR cell therapy. The methods and compositions disclosed herein can be used to suppress or prevent rejection of CAR cells in a human patient. Anti-CD45 ADC can be used to selectively target the lymphocytes of a patient scheduled to receive CAR cell therapy. As described herein, anti-CD45 ADC can also be used to reduce the risk of rejection of CAR cells by targeting and depleting CD45-positive cells in a human patient who has already received CAR cell therapy.

[0118] The compositions and methods described herein can be used to deplete CD45+ cells (e.g., lymphocytes) associated with rejection of CAR cell therapy. The methods of the disclosure promote the acceptance of immune cells expressing CAR in a human subject (e.g., a human subject having cancer or an autoimmune disease). In one embodiment, the method comprises administering an anti-CD45 antibody drug conjugate (ADC) to a human subject scheduled to receive or having received CAR therapy, and administering a therapeutically effective amount of immune cells expressing CAR to the human subject. The CAR-expressing immune cells can be allogeneic or autologous.

[0119] Anti-CD45 ADC can be administered to a human patient in need thereof before, concomitantly with, or after the administration of CAR cell therapy. In one embodiment, anti-CD45 ADC is administered to a human patient in need thereof before the administration of CAR cell therapy (e.g., about 1 day to about 10 days, about 1 day to about 5 days, about 1 day to about 3 days, about 3 days, about 2 days, about 12 It is administered. A single administration of the anti-CD45 ADC can be administered to a human patient either before, after, or simultaneously with the administration of the CAR cell therapy, and such a single administration is sufficient to prevent or reduce the risk of depletion of CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 3 days before the administration of the CAR cell therapy . In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 2 days before the administration of the CAR cell therapy . In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 1 day before the administration of the CAR cell therapy . In one embodiment, the anti-CD 45 ADC is administered to a human patient in need thereof about 20 hours before the administration of the CAR cell therapy . In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 18 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 15 hours before the administration of the C AR cell therapy. In one embodiment , the anti-CD45 ADC is administered to a human patient in need thereof about 12 hours before the administration of the CAR cell therapy and is required. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 6 hours before the administration of the CAR cell therapy . In one embodiment, the anti-CD4 5 ADC is administered to a human patient in need thereof about 4 hours before the administration of the CAR cell therapy and is required. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 2 hours before the administration of the CAR cell therapy and is required. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof simultaneously with the administration of the CAR cell therapy . In one embodiment, the anti-C D45 ADC is administered to a human patient in need thereof about 2 hours after the administration of the CAR cell therapy administered. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 4 hours after administration of the CAR cell therapy. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 6 hours after administration of the CAR cell therapy. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof about 12 hours after administration of the CAR cell therapy. In one embodiment, the anti-CD45 ADC is administered to a human patient in need thereof.

[0120] In one embodiment, the anti-CD45 ADC is administered prior to administration to a human patient in need thereof by CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 1 2 hours to about 21 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 18 hours to about 20 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 20 hours to about 18 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 1 day to about 15 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 1 day to about 10 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 2 days to about 8 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 AD C is administered to a human subject about 1 day to about 15 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD 45 ADC is administered to a human subject about 1 day to about 10 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 2 days to about 8 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to a human subject about 2 days to about 8 days prior to administration of the CAR-expressing immune cells. It is administered. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to the human subject about 3 to about 6 days before the administration of the CAR-expressing immune cells. It is administered. In one embodiment, the anti-CD45 ADC is administered to a human patient in combination with CAR therapy, and the anti-CD45 ADC is administered to the human subject about 3 to about 6 days before the administration of the CAR-expressing immune cells. It is administered.

[0121] In one embodiment, an anti-CD45 ADC with lymphodepleting dose is administered. The overall level of lymphocytes in a biological sample from a human patient can be tested after the administration of the anti-CD45 ADC, where the decrease in the total number of lymphocytes in the human patient after the administration of the anti-CD45 ADC compared to the pre-administration level indicates the effectiveness of the anti-CD45 ADC in preventing rejection of the CAR cell therapy. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is about 5% - 25%, about 5% - 20%, about 5% - 15%, or about 5% - 10% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes is determined within 1 day before the administration of the anti-CD45 ADC. It can be tested after the administration of the anti-CD45 ADC, where the decrease in the total number of lymphocytes in the human patient after the administration of the anti-CD45 ADC compared to the pre-administration level indicates the effectiveness of the anti-CD45 ADC in preventing rejection of the CAR cell therapy. It can be tested after the administration of the anti-CD45 ADC, where the decrease in the total number of lymphocytes in the human patient after the administration of the anti-CD45 ADC compared to the pre-administration level indicates the effectiveness of the anti-CD45 ADC in preventing rejection of the CAR cell therapy. It can be tested after the administration of the anti-CD45 ADC, where the decrease in the total number of lymphocytes in the human patient after the administration of the anti-CD45 ADC compared to the pre-administration level indicates the effectiveness of the anti-CD45 ADC in preventing rejection of the CAR cell therapy. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes in a biological sample from a human patient is at least about 5%, at least about 10%, at least about 15%, or at least about 20% lower compared to the level of lymphocytes in a biological sample from the human patient (the same type of biological sample, such as blood) immediately before the administration of the anti-CD45 ADC. In one embodiment, the level of endogenous lymphocytes is determined within 1 day before the administration of the anti-CD45 ADC.

[0122] The level of lymphocytes is determined according to standard methods known in the art, including but not limited to fluorescence-activated cell sorting (FACS) analysis or a blood analyzer. The level of lymphocytes is determined according to standard methods known in the art, including but not limited to fluorescence-activated cell sorting (FACS) analysis or a blood analyzer. The level of lymphocytes is determined according to standard methods known in the art, including but not limited to fluorescence-activated cell sorting (FACS) analysis or a blood analyzer.

[0123] Normal levels of neutrophils are required to prevent infection. Neutropenia occurs when the level of neutrophils in the blood is abnormally low, leading to an increased susceptibility to infection (see, for example, Schwartzberg, Lee S. “Neutropenia: et iology and pathogenesis.” Clinical corne rstone 8 (2006): S5 - S11. This reference is hereby incorporated by reference in its entirety into this specification.). Neutropenia is often caused by chemotherapy treatment, side effects, or autoimmune diseases. Methods for measuring the absolute neutrophil count (ANC) in a subject's blood are known in the art (see, for example, Amundsen, Erik K. , et al. American journal of clinical pa , et al. American journal of clinical pathology. 137.6 (2012): 862 - 869. This reference is hereby incorporated by reference in its entirety into this specification.).

[0124] In one embodiment of the methods disclosed herein, a human subject does not develop neutropenia after administration of immune cells expressing a CAR. In certain embodiments, neutropenia is defined as a human subject having an absolute neutrophil count (ANC) of less than about 1500 per microliter of blood (e.g., less than about 1500 / μL, less than about 1400 / μ L, less than about 1300 / μL, less than about 1200 / μL, less than about 1100 / μL, less than about 10 00 / μL, less than about 900 / μL, less than about 800 / μL, less than about 700 / μL, or also less than about 600 / μL). Clinically, severe neutropenia is defined as an absolute neutrophil count in the blood of less than 500 / μL

[0125] Neutropenia or agranulocytosis can be a cause of infection and death. In one embodiment the human subject does not develop severe neutropenia after administration of immune cells expressing a CAR. In certain embodiments of the methods disclosed herein, severe neutropenia is defined as an ANC in the blood of less than about 500 / μL (e.g., less than about 500 / μL, less than about 450 / μL, less than about 400 / μ L, less than about 350 / μL, less than about 300 / μL, less than about 250 / μL, less than about 200 / μ L, less than about 150 / μL, or less than about 100 / μL).

[0126] In one embodiment, administration of an ADC (e.g., at a lymphodepleting dose) is effective to increase the level of one or more CAR-T engraftment cytokines (i.e., cytokines that are beneficial for engraftment of CAR-T and are associated with the proliferation and efficacy of CAR-T) in a human subject relative to, for example, a baseline level or relative to the level of one or more CAR-T engraftment cytokines in the human subject prior to administration of the ADC or relative to a predetermined threshold level. In certain embodiments, the level of CAR-T engraftment cytokines is equivalent to the level of CAR-T engraftment cytokines in patients treated with fludarabine / cyclophosphamide conditioning (e.g., see patient data disclosed in Kochehnderfer et al. Clin Oncol. 35:1 803-13). In certain embodiments, the CAR-T engraftment cytokines are IL-15 and / or IL-7 (e.g., see Example 4) . . [[ID= forty]]

[0127] In one embodiment, administration of an anti-CD45 ADC (e.g., in a lymphodepleting dose) is For example, one or more cytokine release syndrome (CRS) events in a human subject relative to a baseline level. -Does not substantially increase cytokine levels. For example, see Lee, Daniel W., et al. Blood. 124 2 (2014):188-195. In certain embodiments, anti-CD Administration of a 45ADC can, for example, reduce one or more CRS in a human subject prior to administration of the ADC. - one or more cytokine levels in a human subject, or relative to a predetermined threshold In certain embodiments, the CR5 inhibitor does not substantially increase the levels of the above CRS-cytokines. S-cytokines include IFNγ, IL-10, IL-6, IL-8, MIP-1α, and MI P-1β, or IL-10.

[0128] In some embodiments, administration of the ADC (e.g., at a lymphocyte-depleting dose) is One or more CAR-T engraftment cytokines (i.e., beneficial for CAR-T engraftment) in the rats and increase levels of cytokines associated with CAR-T proliferation and efficacy. However, it has been shown to be effective against one or more cytokine release syndrome (CRS)-related disorders in human subjects. Does not increase cytokine levels.

[0129] As mentioned above, one advantage of the methods described herein is that they are effective in treating patients undergoing CAR therapy and In a conditioning regimen administered to a human patient planning to receive The amount of lymphodepleting chemotherapy can be reduced or eliminated Yes. Although not limited, lymphodepleting chemotherapeutic agents such as fludarabine, cyclophosphamide, bendamustine, and / or pentostatin are commonly used as resistance inhibitors to promote the acceptance of CAR-expressing cells in humans undergoing CAR therapy. In certain embodiments, a human patient is administered an anti-CD45 ADC in combination with (e.g., prior to) the administration of CAR-expressing immune cells (e.g., T cells), such that the human patient is not administered a lymphodepleting chemotherapeutic agent (e.g., fludarabine and / or cyclophosphamide) before, simultaneously with, or after the administration of the CAR-expressing immune cells. In certain embodiments, the anti-CD45 ADC is used in combination with another therapy to promote the tolerance of CAR-expressing immune cells. Using an anti-CD45 ADC as an agent to deplete endogenous immune cells in a human subject and reduce the risk of rejection of CAR-expressing immune cells can also avoid or reduce the use of other immunosuppressive agents. For example, alemtuzumab is commonly used as a resistance inhibitor in combination with CAR therapy to promote the acceptance of CAR-expressing cells in humans undergoing CAR therapy. In certain embodiments, a human

[0130] patient is administered an anti-CD45 ADC in combination with (e.g., prior to) the administration of CAR-expressing immune cells (e.g., T cells), such that the human patient is not administered alemtuzumab before, simultaneously with, or after the administration of the CAR-expressing immune cells. The methods disclosed herein can be used for both autologous and allogeneic cells expressing a CAR. Importantly, the anti-CD45 ADC conditioning described herein is useful for promoting the acceptance of CAR-expressing cells in humans undergoing CAR therapy. In certain embodiments, a human patient is administered an anti-CD45 ADC in combination with (e.g., prior to) the administration of CAR-expressing immune cells (e.g., T cells), such that the human patient is not administered alemtuzumab before, simultaneously with, or after the administration of the CAR-expressing immune cells. As a result, the human patient is not administered alemtuzumab before, simultaneously with, or after the administration of the CAR-expressing immune cells.

[0131] The methods disclosed herein can be used for both autologous and allogeneic cells expressing a CAR. Importantly, the anti-CD45 ADC conditioning described herein The engrafting method is by providing a means that can bring about tolerance of allogeneic cells and is useful for expanding the types of immune cells that can be used in CAR therapy. In one embodiment the CAR-expressing immune cells are allogeneic cells or autologous cells. Examples of types of immune cells that can be engineered to express CAR include, but are not limited to, allogeneic T cells, autologous T cells, autologous NK cells, or allogeneic NK cells.

[0132] In one embodiment, an anti-CD45 antibody-drug conjugate is used to deplete donor cells (e.g., lymphocytes expressing CD45) that express CD45 by administering the anti-CD45 antibody-drug conjugate after the implementation of CAR cell therapy. In one embodiment the CAR cell therapy includes allogeneic cells.

[0133] The methods disclosed herein are particularly useful for the treatment of cancer or autoimmune diseases in human subjects having one of these disorders.

[0134] In one embodiment, the methods disclosed herein are used to treat cancer. More specifically, an anti-CD45 ADC is administered to a human subject having cancer in combination with CAR therapy. Examples of types of cancer that can be treated using the methods disclosed herein include, but are not limited to adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer , ovarian cancer, triple-negative breast cancer, hematopoietic / lymphoid cancer, liver metastasis of colorectal cancer, small cell lung cancer, non-small cell lung cancer , B-cell lymphoma, relapsed or refractory B-cell lymphoma, follicular lymphoma, mantle cell lymphoma , diffuse large B-cell lymphoma, relapsed or refractory diffuse large B-cell lymphoma ​​​​​Lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, recurrent mediastinal / refractory mediastinal lymphoma Recurrent mediastinal B-cell lymphoma (refractory tory mediastinal large B-cell lymphoma), Large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, relapsed or refractory non-Hodgkin's lymphoma, refractory aggressive non-Hodgkin's lymphoma Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, colorectal carcinoma , gastric cancer, pancreatic carcinoma, triple-negative invasive breast cancer, renal cell carcinoma, squamous cell carcinoma of the lung, hepatocellular carcinoma , urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia Blastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic Leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia Hematologic malignancies, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory Multiple myeloma, multiple myeloma of bone, malignant glioma of the brain, myelodysplastic syndrome, EGFR positive Colorectal cancer, glioblastoma multiforme, neoplasm, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumor tumors, advanced solid tumors, mesothelin-positive tumors, hematologic malignancies, and other advanced malignancies This includes tumors.

[0135] In one embodiment, the methods disclosed herein are used to treat an autoimmune disease. More specifically, anti-CD45 ADCs, in combination with CAR therapy, are being used to treat autoimmune diseases. The combination methods disclosed herein are administered to human subjects having Examples of autoimmune diseases that can be obtained include, but are not limited to, multiple sclerosis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, lupus, and psoriasis.

[0136] In certain embodiments, the anti-CD45 ADC is administered to a human patient in combination with CAR-T cell therapy. In one embodiment, the anti-CD45 ADC is administered to a human patient prior to administration of CAR-T therapy. Examples of CAR-T cells that can be used in combination with the anti-CD45 ADC therapy described herein include, but are not limited to, the following: CD19 CAR-T (e.g., CART-19-01, 02, 03 (Fujian Medical University); daopeicart (Hebei Senlang Biotechnology Inc.); IM19CART / 001, YMCART2 01702 (Beijing Immunochina Medical Scienc e & Technology Co.); CART-CD19-02, 03 (Wuhan Sian Medical Technology Co.); Universal CD19-CART / SHBYCL001, 002 (Shanghai Bioray Laboratory Inc.); UnicarTherapy201701 (Sh anghai Unicar-Therapy Biomedicine Techno logy Co.); Genechem / NCT02672501 (Shanghai GeneChem Co.); SenL_19 (Hebei Senlang Biot echnology Inc.); PCAR-019 (PersonGen BioTh echnology Inc.); PCAR-019 (PersonGen BioTher echnology Inc.); PCAR-019 (PersonGen BioTh erapeutics (Suzhou); ICAR19 (Immune Cell, I nc.); WM-CART-02 (Sinobioway Cell Therapy Co.); HenanCH080,109,152 (Henan Cancer Hos pital / The Pregene (ShenZhen) Biotechnolog y Co.); IM19-CD28 and IM19-41BB CAR-T cells (Bei jing Immunochina Medical Science & Techn ology Company); CTL019 / IT1601-CART19 (Beij ing Sanwater Biological Technology Co.); CTL019 / CCTL019C2201 (Novartis Pharmaceuti cals); CD19:4-1BB:CD28:CD3 / FirstShenzhen0 1 (Shenzhen Second People’s Hospital / The Beijing Pregene Science and Technology C ompany); MB-CART19.1 (Shanghai Children’s Medical Center / Miltenyi Biotec GmbH); PZ0 1 CAR-T cells (Pinze Lifetechnology Co.); YMCA RT201701 (Beijing Immunochina Medical Sci ence & Technology Co.); 2016YJZ12 (Peking University / Marino Biotechnology Co.); EGF Rt / 19-28z / 4-1BBL CAR T cells (Memorial Sloan Kettering Cancer Center / Juno Therapeutic s, Inc.);Doing-002(Beijing Doing Biomedi cal Co.);PCAR-019(PersonGen BioTherapeut ics(Suzhou) Co.);C-CAR011(Peking Union M edical College Hospital / Cellular Biomedi cine Group Ltd.);iPD1 CD19 eCAR T cells(Peki ng University / Marino Biotechnology Co.); 2013-1018 / NCT02529813(M.D. Anderson Canc er Center / Ziopharm / Intrexon Corp.);Henan CH CAR 2-1(Henan Cancer Hospital / The Pre gene(ShenZhen) Biotechnology Co.);JCAR01 5(Juno Therapeutics,Inc.);JCAR017 / 017001 ,004,006(Juno Therapeutics, Inc.);JCAR01 7(Celgene);TBI-1501(Takara Bio Inc.);JMU -CD19CAR(Autonomous Medical University);KTE-C19(Kite, A Gilead Company);TriCAR-T-CD19(Timmune Biotech I nc.);PF-05175157(Fred Hutchinson Cancer [[ID=;CD22 / CD30 / CD7 / BCMA / CD1 23(e.g.,2016040 / NCT03121625(Hebei Senlan g Biotechnology Inc.); CD22 (e.g., Ruijin-CAR -01 (Ruijin Hospital / Shanghai Unicar-Ther apy Bio-medicine Technology Co.); AUTO-PA 1, DB1 (Autolus Limited)), CD20 (e.g., Doing-006 (Beijing Doing Biomedical Co.)); or CD20 / CD22 / CD30 (e.g., SZ5601 (The First Affiliated Hospital of Soochow University Shanghai / Unicar-Therapy Bio-medicine Technology C o.)).

[0137] CAR construct

[0138] This disclosure includes the use of CAR therapy in combination with anti-CD45 immunosuppressive ADC. This disclosure is generally not limited to a specific CAR construct, e.g., a specific antigen-binding region or intracellular sig nal transduction domain. Because this disclosure is based on the discovery that anti-CD45 ADC can function as a conditioning agent for CAR therapy by removing endogenous CD45+ immune cells such as endogenous lymphocytes, thereby promoting the acceptance of CAR-expressing cells by promoting the acceptance of CAR-expressing cells and thus promoting the acceptance of CAR-expressing cells . Specific CARs (e.g., CD19-specific CAR) are contemplated in this specification and are included in the methods disclosed herein, but are not intended to be limiting in this specification and are included in the methods disclosed herein, but are not intended to be limiting .

[0139] CAR constructs are known in the art and generally comprise (a) an antigen-binding domain An extracellular region containing ン, (b) a transmembrane domain, and (c) a cytoplasmic signaling domain including. Exemplary CAR configurations are known in the art, and any suitable configuration can be used in the methods described herein . For example, the CAR can be, for example, as described in Guedan et al. Molecular Therapy-Methods & Clinical Dev elopment. 12:145-156 (2019) or Sadelain et al. Cancer discovery 3.4:388-398 (201 3), and can be a first-generation, second-generation, or third-generation CAR, as described in , and the entire contents of which are incorporated herein by reference. Briefly, a "first-generation" CAR can include (a) an extracellular antigen-binding domain, (b) a transmembrane domain, (c) one or more intracellular signaling domains, and optionally (d) a hinge region that connects the antigen-binding domain to the transmembrane domain . A "second-generation" CAR can include elements (a), (b ), (c), and optionally (d), and further includes a co-stimulatory domain (e.g., the co-stimulatory domain of CD28 or 4-1BB). A "third-generation" CAR can include elements( a), (b), (c), and optionally (d), and further includes a plurality of co-stimulatory do mains (e.g., the co-stimulatory domains of CD28 and 4-1BB, or CD28 and O X40 co-stimulatory domains). Each of the foregoing elements will be described in detail below. In some embodiments, it should be understood that the CAR molecules described by the following exemplary non-limiting arrangements are from left to right from the N-terminus to the C-terminus of the CAR. The CARs described by the present disclosure may include any other combination of elements as described herein , may further be included. Other exemplary chimeric antigen receptor constructs are described in U.S. Patent No. 9 ,328,156, U.S. Patent No. 9,783,591, U.S. Patent No. 9,714,278 , U.S. Patent No. 9,765,156, U.S. Patent No. 10,117,896, U.S. Patent No. 9,573,988, U.S. Patent No. 10,308,717, U.S. Patent No. 10,221, 245, U.S. Patent No. 10,040,865, U.S. Patent Publication No. 2018 / 025671 2A1, U.S. Patent Publication No. 2018 / 0271907A1, U.S. Patent Publication No. 2016 / 0046724A1, U.S. Patent Publication No. 2018 / 0044424A1, U.S. Patent Publication No. 2018 / 0258149A1, U.S. Patent Publication No. 2019 / 0151363A1, and U.S. Patent Publication No. 2018 / 0273601A1, and the contents of each of the foregoing patents and patent publications are hereby incorporated by reference in their entirety.

[0140] The CARs used in the methods disclosed herein include an extracellular antigen-binding domain. The extracellular antigen-binding domain can be any molecule that binds to an antigen and includes, but is not limited to, human antibodies, humanized antibodies, or any functional fragment thereof. In certain embodiments, the antigen-binding domain is a scFv. In other embodiments, the extracellular antigen-binding domain is a non-immunoglobulin scaffold protein. In other embodiments, the extracellular binding domain of the CAR includes a single-chain T cell receptor (scTCR). As described in U.S. Patent No. 5,359,046, U.S. Patent No. 5,686,281, and U.S. Patent No. 6,103, 521, the extracellular domain can mediate ligand binding and / or signal Obtained from any of a variety of extracellular domains or secreted proteins related to signal transduction It can also be done.

[0141] The selection of the molecular target (antigen) of the extracellular binding domain depends on the type and number of ligands that define the surface of the target cell For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, It can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, in one aspect, by engineering an extracellular antigen-binding domain that specifically binds to a desired antigen and incorporating it into a CAR, the response of immune cells (e.g., T cells) via the CAR can be directed to the antigen of interest. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state such as cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain of a CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, In some embodiments, the CAR is engineered to target a desired tumor antigen by engineering an antigen-binding domain that specifically binds to an antigen on tumor cells. In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, In the context of the present disclosure, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1 CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, , CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123 is included. In one embodiment, the CAR is CD19, CD22, CD30, C D7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, meso thyrin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA , TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, N KR-2, PSCA, CD28, TAA, NKG2D or CD123 binds to the scF v is included.

[0142] In one embodiment, as described in U.S. Patent Application Publication No. 20190388528 (Bluebird Bio), the CAR binds to BCMA, and the content thereof related to the CAR is incorporated herein by reference.

[0143] In another aspect, the extracellular binding domain of the CAR binds to an antigen, and the antigen is AFP ( e.g., ETCH17AFPCAR01 (Aeon Therapeutics (Shang hai) Co. / Eureka Therapeutics Inc.)), GPC3 (e.g., GeneChem GPC-3 CART (Shanghai GeneChem Co.), 302 GPC3-CART (Shanghai GeneChem C o.); CAR-T for liver cancer (Shanghai GeneChem Co.), CAR-GPC3 T cells (Carsgen Therapeutics)), MUC1 (e.g., PG-021-001,002 (PersonGen BioTherapeut ics (Suzhou) Co.)), mesothelin (e.g., H2017-01-P01 (N ingbo Cancer Hospital);TAI-meso-CART(Sha nghai GeneChem Co.);K16-4 / NCT02930993(Ch ina Meitan General Hospital / Marino Biote chnology Co.))、CD38(eg: Anti-CD38 A2 CAR-T / SOR-CART-MM-001(Sorrento Therapeutics, Inc.))、herinCAR-PD1(eg: herinCAR-PD1 / NBWYK Y2016-06-001,002,003(Ningbo Cancer Hospi tal);SIMC-20160101,02,03(Shanghai Intern ational Medical Center))、BCMA(eg: P-BCMA-1 01 autologous T stem cell memory(Tscm)CA R-T cells / P-BCMA-101-001(Poseida Therapeutic s, Inc.);HenanCH284(Henan Cancer Hospita l / The Pregene (ShenZhen) Biotechnology Company); LCAR-B38M CAR-T cells(Nanjing Leg end Biotech Co.);9762 / NCT03338972(Fred H utchinson Cancer Research Center / Juno Th erapeutics, Inc.);Descartes-08(Cartesian Therapeutics);KITE-585(Kite, A Gilead C ompany);bb21217(bluebird bio);bb21217(Ce lgene); JCARH125 (Juno Therapeutics, Inc.) )、CD30 (e.g., ICAR30 T cells (Immune Cell, Inc.))、 EGFR (e.g., EGFR:4-1BB:CD28:CD3 modified T cells / First Sh enzhen02 (Shenzhen Sceond People’s Hospit al / The Beijing Pregene Science and Techn ology Company); EGFR-IL12-CART (Shenzhen S econd People’s Hospital / The Pregene (Shen Zhen) Biotechnology Co.); SBNK-2016-015-0 1 (Beijing Sanbo Brain Hospital / Marino Bi otechnology Co.))、MG7 (e.g., MG7-CART (Xijing Hospital / Shanghai GeneChem Co.))、BCMA / TA CI (e.g., AUTO2-MM1 (Autolus Limited))、CEA (e.g., 3 83-74 / NCT02416466 (Roger Williams Medica l Center / Sirtex Medical))、Mesothelin / PSCA / C EA / HER2 / MUC1 / EGFRvIII (e.g., NCT03267173 (Firs t Affiliated Hospital of Harbin Medical University / Shanghai Unicar-Therapy Bio-m edicine Technology Co.))、CD20 (e.g., EY201605 It should be noted that there seem to be some unclear or potentially incorrect expressions in the original text, especially in parts like "lgene", "Sceond", "Pregene(Shen Zhen)", etc. which might need further clarification for more accurate understanding and translation.-19 (Beijing Biohealthcare Biotechnology Co.)), CD33 (e.g., 2016-0341 / NCT03126864 (M.D. Anderson Cancer Center / Intrexon Corp. / Zi opharm)), EGFR / BCMA (e.g., EGFRt / BCMA-41BBz CA R T cells (Memorial Sloan Kettering Cancer Ce nter / Juno Therapeutics, Inc.)), ROR2 (e.g., au tologous CCT301-38 or CCT301-59 T cells (Shang hai Sinobioway Sunterra Biotech)), NKR-2( e.g., CYAD-N2T-002,003,004 (Celyad)), PSCA (e.g., B P-012 (Bellicum Pharmaceuticals)), CD28 (e.g., autologous CSR T cells (Beijing Sanbo Brain Hospital / M arino Biotechnology Co.)), TAA (e.g., AMG 119( Amgen)), NKG2D (e.g., CM-CS1 (Celyad)), or CD123 (e.g., UCART123 (Cellectis S.A.)). The foregoing text further provides examples of CARs that bind to said antigen (e.g., AMG119 (Amgen)). These CAR constructs, together with anti-CD45 ADC, can be used in the conditioning methods disclosed herein.

[0144] The CAR construct connects an extracellular antigen-binding domain and a cytoplasmic signaling domain with a transmembrane domain (either literally or by general proximity, e.g., with a spacer).​ further comprises. Generally, a CAR comprises a scFv, Fab or other antibody portion, and generally , can have a hinge or other linker between the scFv (or extracellular antigen-binding domain) and the transmembrane domain. The transmembrane domain binds to an intracellular signaling domain such as CD28 or CD3-ζ and typically includes one or more co-stimulatory domains as described below. domain. In many cases, a spacer or hinge is introduced between the extracellular antigen-binding domain and the transmembrane domain, providing flexibility to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition and binding. domain. In many cases, a spacer or hinge is introduced between the extracellular antigen-binding domain and the transmembrane domain, providing flexibility to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition and binding. domain. In many cases, a spacer or hinge is introduced between the extracellular antigen-binding domain and the transmembrane domain, providing flexibility to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition and binding. domain. In many cases, a spacer or hinge is introduced between the extracellular antigen-binding domain and the transmembrane domain, providing flexibility to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition and binding.

[0145] Thus, in certain embodiments, a CAR can further comprise a hinge region. The hinge region can be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, Ig M, CD28, or CD8 alpha. In one particular embodiment , the hinge region is derived from the hinge region of IgG4. In another embodiment, the hinge of the CAR between the extracellular binding domain and the transmembrane domain is the CD8 hinge domain (see Swis sProt / GenBank accession number P01732). sProt / GenBank accession number P01732). sProt / GenBank accession number P01732).

[0146] In one embodiment, the CAR comprises an extracellular antigen-binding domain and a transmembrane domain connected via the CD8 hinge: AKPTTTPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFA (SEQ ID NO: 9). In one embodiment, the CAR comprises an extracellular antigen-binding domain and a transmembrane domain connected via the CD8 hinge: AKPTTTPAPRPPTPAPTIA

[0147] In one embodiment, the CAR comprises a hybrid CD8-CD28 hinge: AKPTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPR KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP comprises an extracellular antigen-binding domain and a transmembrane domain connected via (Accession No. 10). and.

[0148] The transmembrane domain may be contributed by a protein that contributes to the extracellular antigen-binding domain, an effector function protein that contributes to the signal transduction domain, a protein that contributes to the growth signal transduction portion, or a completely different protein. In most cases, it would be convenient if the transmembrane domain is naturally associated with one of the other domains of the CAR. In one embodiment the transmembrane domain and cytoplasmic domain used are contiguous portions of the CD28 sequence such that any transmembrane domain is contemplated for use herein as long as it can anchor the CAR containing the antigen-binding domain to the cell membrane. The transmembrane domain may be derived from either a natural source or a synthetic source. In the case of a natural source, the domain may be derived from any membrane-bound or transmembrane protein. Particularly used transmembrane domains in the present disclosure are the alpha, beta, or zeta chains of the T cell receptor, CD2 8, CD3 epsilon, CD45, CD4, CD2, CD8, CD9, CD16, CD2

[0149] 2, CD33, CD37, CD64, CD80, CD86, CD134, CD137, C D154, the LFA-1 T cell coreceptor, the CD2 T cell coreceptor / adhesion molecule, the CD8 alpha chain, and fragments thereof (e.g., containing at least the transmembrane domain of these). The transmembrane domain may be any known in the art or described herein ​​​​It can be identified using a method, for example, by using the UniProt database. It is possible.

[0150] In some embodiments, the transmembrane domain can be synthetic, and in that case, the transmembrane domain contains mainly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligo or polypeptide linker, preferably one having a length of 2 to 10 amino acids, can form a bond between the transmembrane domain of the CAR and the cytoplasmic signaling domain. A glycine-serine doublet provides a particularly suitable linker.

[0151] In some embodiments, the transmembrane domain of the CAR of the present disclosure is the CD8 transmembrane domain. The sequence of CD8 for this purpose is taught in PCT Publication No. W02014 / 055771.

[0152] In some embodiments, the transmembrane domain of the CAR is the CD8 transmembrane domain or a functional portion thereof. For example, the CAR can include a CD3 transmembrane domain having the amino acid sequence of LDPKLCYLLDGILFIYGVILT ALFLRVK (SEQ ID NO: 11), or a functional portion thereof such as LCYLLDGILFIYGVILTALFL (SEQ ID NO: 12).

[0153] In some embodiments, the transmembrane domain of the CAR of the present disclosure is the CD28 transmembrane domain. Exemplary sequences of CD28 and exemplary transmembrane domain sequences are provided below. It can be achieved. In some embodiments, the CD28 transmembrane domain is the following exemplary transmembrane domain sequence, or a fragment or variant thereof that can immobilize the CAR containing that sequence on the cell membrane. Thus, in some embodiments, the transmembrane domain of the CAR contains the following amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 13) of the CD28 transmembrane domain. In one embodiment, the transmembrane domain of the CAR contains the following amino acid sequence: IEVMYPPPYLDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF WV (SEQ ID NO: 14) of the CD28 transmembrane domain, or a functional fragment thereof ( for example, SEQ ID NO: 13).

[0154] In addition to the extracellular antigen-binding domain and the transmembrane domain, the CAR further includes an intracellular (or cytoplasmic) signaling domain.

[0155] Signals generated only through the endogenous TCR are insufficient for complete activation of T cells and it is known that secondary or co-stimulatory signals may also be required. In this way, activation of T cells can be mediated by two different classes of cytoplasmic signaling sequences: namely, those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0156] As used herein, "intracellular signaling domain" or "cytoplasmic signaling domain" "Main" refers to the intracellular portion of the molecule. The intracellular signaling domain can generate signals that promote the immune effector function of CAR-containing immune cells (e.g., CAR-T cells or CAR-expressing NK cells). For example, in CART cells or CAR-expressing NK cells the examples of immune effector functions include cytolytic activity and helper activity (including the secretion of cytokines ). In embodiments, the intracellular signaling domain transmits effector -function signals and instructs the cell to perform special functions. Although the entire intracellular signaling domain can be used, often it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain as long as it transmits the effector -function signal. Thus, the term intracellular signaling domain means any truncated portion of the intracellular signaling domain sufficient to transmit the effector -function signal. In one embodiment, the intracellular signaling domain of the CAR comprises the CD 3 zeta signaling region set forth in SEQ ID NO: 15, or the signaling portion thereof. The cytoplasmic signaling domain includes, but is not limited to, CD3 zeta, FcR gamma

[0157]

Chemical formula

[0158] The cytoplasmic signaling domain includes, but is not limited to, CD3 zeta, FcR gamma , FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD2 2, CD79a, CD79b, CD278 ("ICOS"), Fc.epsilon.RI, It may further include those derived from CD66d, DAP10, and DAP12.

[0159] The CAR may further include an "intracellular co-stimulatory domain", and the "intracellular co-stimulatory domain" is , a polypeptide chain derived from the intracellular signaling domain of one or more co-stimulatory proteins such as CD28 and 4-1BB that enhance cytokine production.

[0160] Exemplary co-stimulatory signaling regions include 4-1BB, CD21, CD28, CD27, C D127, ICOS, IL-15Rα, and OX40.

[0161] In certain embodiments, the cytoplasmic co-stimulatory domain of the CAR is the 4-1BB signaling dom ain alone or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR . 4-1BB is a member of the TNFR superfamily having the amino acid sequence provided as GenBank accession number AAA6247 8.2 or equivalent residues derived from non-human species (e.g., mouse, rodent, monkey, ape, etc.); the "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues derived from non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0162] In one embodiment, the intracellular co-stimulatory signaling domain of the CAR is the 4-1BB (CD1 37) co-stimulatory signaling region, or the signaling portion thereof:

Chemical formula

[0163] In one embodiment, the co-stimulatory signaling domain of the CAR is the CD28 co-stimulatory signaling domain, and the sequence of the CAR is as follows: Intracellular domain: CD28 co-stimulatory signaling domain, or a signaling portion thereof:

Chemical formula

[0164] Thus, the cytoplasmic domain of the CAR may include a CD3-zeta signaling domain combined with any other desired cytoplasmic domain(s) useful in the context of the CARs of the present disclosure. In certain embodiments, the cytoplasmic domain of the CAR may include a CD3 zeta domain and a co-stimulatory signaling region including, but not limited to, 4-1BB, CD28, and CD27.

[0165] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CARs of the present disclosure may be linked to each other randomly or in a specific order. Optionally, short oligos or polypeptide linkers or spacers, preferably those having a length of 5 to 20 amino acids, may be inserted between the cytoplasmic domains. GGGGS (SEQ ID NO: 18) or (GGGGS)×3 (SEQ ID NO: 19 ) provide particularly suitable linkers.

[0166] In one embodiment, the CAR used herein includes an extracellular domain comprising a single-chain variable domain of an anti-CD19 monoclonal antibody, a transmembrane domain comprising the hinge and transmembrane domains of CD8α, and a signaling domain of CD3ζ and a signaling ​It includes a cytoplasmic domain containing a transduction domain. Exemplary CARs include the anti-CD19 monoclonal antibody described in Nicholson I C, et al., Mol Immunol 34:1157-1165 (19 97) for the extracellular domain, and the 2 1 amino acid signal peptide (translated from 63 nucleotides at positions 26-88 of GenBank accession number NM_001768 ). The CD8α hinge and transmembrane domains consist of 69 amino acids translated from 207 nucleotides at positions 815-1 021 of GenBank accession number NM_001768. The CD3ζ signaling domain of the preferred embodiment contains 112 amino acids translated from 339 nucleotides at positions 1022-1360 of GenBank accession number NM_00073 4. .

[0167] A spacer or hinge domain can be incorporated between the extracellular domain (including the antigen-binding domain) and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligo or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or cytoplasmic domain in a polypeptide chain. As used herein, the hinge domain generally refers to any oligo or polypeptide that functions to confer flexibility to the CAR or its domain and / or to prevent steric hindrance of the CAR or its domain. In some embodiments, the spacer or hinge domain can be up to 300 amino acids, preferably 10-100 amino acids. amino acids. and / or to prevent steric hindrance of the CAR or its domain. In some embodiments, the spacer or hinge domain can be up to 300 amino acids, preferably 10-100 amino acids. The amino acids may include, most preferably, 5 to 20 amino acids. Also, aspects of the present disclosure are not limited in this regard, and it should be understood that one or more spacer domains may be included in other regions of the CAR.

[0168] The CAR may include a region having the sequences provided herein (e.g., an antigen-binding domain, a transmembrane domain, a cytoplasmic domain, a signaling domain, a safety domain, and / or a linker, or any combination thereof) or a variant thereof, or a fragment of any one of them (e.g., a variant and / or fragment that retains the functions necessary for CAR activity) may be included in the CAR proteins described herein. In some embodiments, the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes relative to the exemplified sequences. In some embodiments, the variant has a sequence that is at least 80%, at least 85%, at least 90%, 9 0% - 95%, at least 95% or at least 99% identical to the exemplified sequences. In some embodiments, the fragment is 1 - 5, 5 - 1 0, 10 - 20, 20 - 30, 30 - 40 or 40 - 50 amino acids shorter than the sequences provided herein. In some embodiments, the fragment is shorter in the N-terminal, C-terminal or both terminal regions of the provided sequence. In some embodiments, the fragment comprises 80% - 85%, 85% - 90%, 90% - 95% or 95% - 9 9% of the number of amino acids in the sequences provided herein.

[0169] In other embodiments, the present disclosure includes nucleic acid sequences encoding the amino acid sequences disclosed herein.​​​

[0170] In some embodiments, the exemplary non-limiting arrangement described above is from left to right, from the N-terminus to the C-terminus of the CAR. The CAR may comprise any other combination of the elements described herein, or may further comprise them. from the N-terminus to the C-terminus of the CAR. The CAR may comprise any other combination of the elements described herein, or may further comprise them. include, or may further include.

[0171] Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population). Once the CAR construct is specified in its various parts, CAR-expressing immune cells are produced and the immune cells come to express the CAR. This method involves introducing (e.g., transducing) into the immune cells a nucleic acid molecule described herein (e.g., an RNA molecule (such as mRNA)), or a vector comprising a nucleic acid molecule encoding the CAR described herein. The present disclosure also provides a method of generating a population of cells (e.g., RNA-engineered cells that transiently express exogenous RNA). This method involves introducing into the cells an RNA described herein (e.g., in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding the CAR polypeptide described herein). In an embodiment, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cells are the cells described herein, e.g., immune effector cells (e.g., T cells or NK cells, or a cell population).

[0172] CAR-expressing immune cells can be administered as a dose based on the number of cells per kilogram of body weight of the subject to which the cells are administered (cells / kg). For example, in some embodiments, the subject is administered from about 1×10 to about 1×10 cells / kg (e.g., from about 1×10 6 to about 2×10 8 cells / kg (e.g., from about 1×10 6 to about 2×10 6 ), About 2×10 6 ~About 3×10 6 、About 3×10 6 ~About 4×10 6 、About 4×10 6 ~About 5×10 6 、About 5×10 6 ~About 6×10 6 、About 6×10 6 ~About 7×10 6 、About 7×10 6 ~About 8× 10 6 、About 8×10 6 ~About 9×10 6 、About 9×10 6 ~About 1×10 7 、About 1×10 7 ~About 2×10 7 、About 2×10 7 ~About 3×10 7 、About 3×10 7 ~About 4×10 7 、About 4×10 7 ~About 5×10 7 、About 5×10 7 ~About 6×10 7 、About 6×10 7 ~About 76×10 7 、About 8× 10 7 ~About 9×10 7 、About 9×10 7 ~About 1×10 8 、About 1×10 6 、About 1×10 7 、Or Or about 1×10 8 cells / kg) are administered. In one embodiment, the subject is about 1×10 6 ~ about 2×10 6 engineered CAR T cells per kg (e.g., about 1×10 6 、About 1.1 ×10 6 、About 1.2×10 6 、About 1.3×10 6 、About 1.4×10 6 、About 1.5×10 6 、About 1.6×106 , about 1.7×10 6 , about 1.8×10 6 , or about 1.9×10 6 cells / kg) is administered.

[0173] In some embodiments, the dosage of CAR-expressing immune cells depends on the mode and location of administration and is in the range of about 10 4 to about 10 10 cells / kg of body weight, for example, about 10 5 to about 10 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 , or about 10 5 to 10 6 . Generally, for systemic administration , a higher dosage is used than for local administration where the immune cells of the present invention are administered to the tumor region . Exemplary dosage ranges include, but are not limited to, 1×10 4 to 1×10 8 , 2× 10 4 to 1×10 8 , 3×10 4 to 1×10 8 , 4×10 4 to 1×10 8 , 5×10 4 to 1×10 8 , 6×10 4 to 1×10 8 , 7×10 4 to 1×10 8 , 8×10 4 to 1×10 8 , 9×10 4 to 1×10 8 , 1×10 5 to 1×10 8 , for example, 1×10 5 to 9×10 7 , 1×10 5 to 8×10 7 , 1×10 5~7×10 7 、1×10 5 ~6×10 7 、1× 10 5 ~5×10 7 、1×10 5 ~4×10 7 、1×10 5 ~3×10 7 、1×10 5 ~ 2×10 7 、1×10 5 ~1×10 7 、1×10 5 ~9×10 6 、1×10 5 ~8×10 6 、1×10 5 ~7×10 6 、1×10 5 ~6×10 6 、1×10 5 ~5×10 6 、1× 10 5 ~4×10 6 、1×10 5 ~3×10 6 、1×10 5 ~2×10 6 、1×10 5 ~ 1×10 6 、2×10 5 ~9×10 7 、2×10 5 ~8×10 7 、2×10 5 ~7×10 7 、2×10 5 ~6×10 7 、2×10 5 ~5×10 7 、2×10 5 ~4×10 7 、2× 10 5 ~3×10 7 、2×10 5 ~2×10 7 、2×10 5 ~1×10 7 、2×10 5 ~ 9×10 6 、2×10 5 ~8×10 6 、2×10 5 ~7×10 6 、2×10 5 ~6×10 6 、2×10 5 ~5×10 6 、2×10 5 ~4×10 6 、3×10 5 ~3×10 6 cells / kg, etc. are included. Also, exemplary dosage ranges include, but are not limited to, 5×1 0 5 ~1×10 8 、for example, 6×10 5 ~1×10 8 、7×10 5 ~1×10 8 、8×1 0 5 ~1×10 8 、9×10 5 ~1×10 8 、1×10 6 ~1×10 8 、1×10 6 ~9 ×10 7 、1×10 6 ~8×10 7 、1×10 6 ~7×10 7 、1×10 6 ~6×10 7 、1×10 6 ~5×10 7 、1×10 6 ~4×10 7 、1×10 6 ~3×10 7 cells / k g, etc. may also be included. Exemplary cell dosages include, but are not limited to, about 10 4 ~about 10 10 cells / kg in the range of about 1×10 4 、about 2×10 4 、about 3×10 4 、about 4×104 , about 5×10 4 , about 6×10 4 , about 7×10 4 , about 8×10 4 , about 9×10 4 , about 1×1 0 5 , about 2×10 5 , about 3×10 5 , about 4×10 5 , about 5×10 5 , about 6×10 5 , about 7 ×10 5 , about 8×10 5 , about 9×"10 5 , about 1×10 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 6×10 6 , about 7×10 6 , about 8×10 6 , about 9×10 6 , about 1×10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6× 10 7 , about 7×10 7 , about 8×10 7 , about 9×10 7 , about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 , about 6×10 8 , about 7×10 8 , about 8×10 8 , about 9×10 8 , about 1×10 9 Doses such as about 1×10 cells / kg are included.

[0174] In some embodiments, the dose of CAR-expressing immune cells (e.g., CAR-T cells) is a non- weight-based determination and is instead based on the total number of cells administered. For example, in some embodiments, the subject is administered a total dose of about 1×10 7 to about 9×10 8 cells (e.g., about 1×10 7 to about 9 ×10 8 cells, about 1×10 7 to about 8×10 8 cells, about 1×10 7 to about 7×10 8 cells, about 1×10 7 to about 6×10 8 cells, about 1×10 7 to about 5×10 8 cells, about 1×10 7 to about 4 ×10 8 cells, about 1×10 7 to about 3×10 8 cells, about 1×10 7 to about 2×10 8 cells, about 1×10 7 to about 1×10 8 cells, about 2×10 7 to about 9×10 8 cells, about 3×10 7 to about 8 ×10 8 cells, about 4×10 7 to about 7×10 8 cells, about 5×10 7 to about 6×10 8 cells, about 6×10 7 to about 6×10 8 cells). In some embodiments, the subject is administered no more than about 9×10 8 cells (e.g., no more than about 9×10 8 cells, no more than about 8×10 8 cells, no more than about 7×10 8 cells, no more than about 6×10 8 cells, no more than about 5×10 8 cells, no more than about 4×10 8Hereinafter, about 3×10 8 Hereinafter, about 2×10 8 Hereinafter, about 1×10 8 Hereinafter, about 9×10 7 Hereinafter, about 8×10 7 Hereinafter, about 7×1 0 7 Hereinafter, about 6×10 7 Hereinafter, about 5×10 7 Hereinafter, about 4×10 7 Hereinafter, about 3×10 7 Hereinafter , about 2×10 7 Hereinafter, or about 1×10 7 Hereinafter, the total dose of the cells) is administered.

[0175] In one embodiment, the CAR-expressing immune cells are axicabtagene ciloleucel, which is a CD19-directed gene-modified autologous T cell immunotherapy method. Therefore, in some embodiments, the subject is pre-treated with a lymphodepleting dose of an anti-CD 45 antibody-drug conjugate (ADC) before administration (e.g., by infusion) of an effective therapeutic amount of axicabtagene ciloleucel, and the anti-CD45 ADC comprises an anti-CD45 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker . In one embodiment, the subject is pre-medicated with acetaminophen (e.g., 650 mg oral administration) and an H1-antihistamine agent (e.g., diphenhydramine 12.5 mg intravenous or oral administration) about 1 hour before administration of axicabtagene ciloleucel. In certain embodiments, the subject is not administered a systemic corticosteroid. In some embodiments, the administration of axicabtagene ciloleucel is based on the number of chimeric antigen receptor (CAR) positive viable T cells. In certain embodiments, the subject is per kilogram of body weight

[0176] (CAR) positive viable T cells. In certain embodiments, the subject is per kilogram of body weight or 2×10 6 The dose of axicabtagene ciloleucel comprising 2×10 positive viable T cells is administered, with a maximum of 2×10 8 positive viable T cells being administered.

[0177] In some embodiments, the subject to whom axicabtagene ciloleucel is administered is a recurrent or refractory adult subject with large B-cell lymphoma. In certain embodiments, the B cell lymphoma is diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma. In some embodiments, the subject has previously received two or more lines of systemic therapy. In some embodiments, the subject does not have primary central nervous system lymphoma.

[0178] In another embodiment, the human subject is not administered a lymphodepleting chemotherapeutic agent, such as fludarabine or cyclophosphamide, prior to administration of axicabtagene ciloleucel.

[0179] In one embodiment, the CAR-expressing immune cells are tisagenlecleucel, a CD19-directed gene-modified autologous T cell immunotherapy method. Thus, in some embodiments, the subject is pre-treated with a lymphodepleting dose of an anti-CD45 antibody-drug conjugate ( ADC), for example, by infusion, prior to administration of a therapeutically effective amount of tisagenlecleucel, where the anti-CD45 ADC comprises an anti-CD45 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker. In one embodiment, the subject is administered acetaminophen approximately 30 to 60 minutes before administration of tisagenlecleucel. ​ and premedicated with an H1 - antihistamine agent (e.g., diphenhydramine). In certain embodiments, the subject is not administered a systemic corticosteroid.

[0180] In some embodiments, the administration of tisagenlecleucel is based on the number of chimeric antigen receptor (CAR) - positive viable T cells. In certain embodiments, the subject has pediatric or young adult B - cell ALL and is up to 25 years old. In some such embodiments, a subject having pediatric or young adult B - cell ALL is administered intravenously a dose of tisagenlecleucel containing (i) 0.2 - 5.0×10 6 CAR - positive viable T cells per kg of body weight if the subject's body weight is 50 kg or less, or (ii) 0.1 - 2.5×10 8 total CAR - positive viable T cells (non - weight - based) if the patient's body weight is over 50 kg.

[0181] In some embodiments, a subject administered tisagenlecleucel is an adult subject having relapsed or refractory large B - cell lymphoma. In some embodiments, an adult subject having relapsed or refractory diffuse large B - cell lymphoma (adult relapsed or refractory diffuse large B - cell lymph oma) is administered intravenously a dose of tisagenlecleucel containing 0.6 - 6.0×10 8 CAR - positive viable T cells. In certain embodiments, the B - cell lymphoma is diffuse large B - cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B - cell lymphoma, high - grade B - cell lymphoma, and DLBCL arising from follicular lymphoma. It is L. In some embodiments, the subject has previously received systemic therapy of two or more lines. There is. In some embodiments, the subject does not have primary central nervous system lymphoma.

[0182] In another embodiment, the human subject is not administered a lymphocyte-depleting chemotherapeutic agent such as fludarabine, cyclophosphamide or bendamustine before administration of tisagenlecleucel. .

[0183] The dose of CAR-expressing immune cells is also adjusted considering whether it is a single administration or multiple administrations. The exact determination of what is considered an effective dose can be based on factors specific to each subject, including the size, age, sex, weight and condition of the particular subject, as described above. The dose can be readily determined by one of ordinary skill in the art based on the disclosure herein and the knowledge of the art. Obtainable.

[0184] The administration of CAR-expressing immune cells to a subject can be carried out by any suitable method. In some embodiments, the cells are administered subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously (e.g., by injection), or intraperitoneally to the patient. In one embodiment, the cells are administered to the patient by subcutaneous injection. In another embodiment, the cells are administered intravenously. In certain embodiments, the cells can be directly injected into a tumor, lymph node, or site of infection. Optionally, a proliferative agent and / or a differentiating agent can be administered to the subject before, during, or after administration of the cells to increase in vivo production of the cells.

[0185] III. Anti-CD45 antibody-drug conjugate (ADC)

[0186] ​As described herein, anti-CD45 ADCs can be used to treat cancer or autoimmune diseases in human patients. More specifically, anti-CD4 5ADCs are directed against CD45+ cells (e.g., CD45+ receptors) in human subjects who are also receiving CAR therapy. Anti-CD45 ADCs can be used to target endogenous lymphocytes and deplete endogenous lymphocytes. These cells are then killed, allowing the patient's immune system to react with the CAR-expressing immune cells (autologous) administered to the subject. Therefore, anti-CD45 ADCs are effective in preventing CAR therapy. used as a conditioning step in combination with the method to induce the expression of engineered CAR-expressing immunoglobulins. Promotes immune cell acceptance in recipient patients. One advantage of using anti-CD45 ADCs is that they can be administered with common lymphodepleting chemotherapy agents. Compared with conventional CAR therapy conditioning methods that involve administering CD45 to endogenous The advantage of this approach is that it allows for the specific targeting of depletion of inflammatory lymphocytes.

[0187] Anti-CD45 antibody

[0188] ADCs that can bind to CD45 may prevent or mitigate the risk of rejection of CAR-expressing immune cells. By reducing the It can be used as a therapeutic agent.

[0189] The anti-CD45 ADCs described herein are anti-CD45 antibodies or their derivatives linked to a cytotoxin. The antigen-binding portion of

[0190] CD45 is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase that binds to T cells. CD45 is essential for signaling through the CD45 and B cell antigen receptors. It includes an extracellular domain and a cytoplasmic domain containing phosphatase. CD45 acts as both a positive and a negative regulator depending on the nature of the stimulus and the type of cells involved. There are a number of possible permutations in the CD45 gene although only six isoforms have been conventionally identified in humans. The isoforms are RA (Uniprot accession number: P08575-8; SEQ ID NO: 20), RO (NCBI accession number: NP_563578.2; SEQ ID NO: 21), RB ( NCBI accession number: XP_006711537.1; SEQ ID NO: 22), RAB (NCBI accession number: XP_006711535.1; SEQ ID NO: 23), RBC (NCBI accession number: XP_006711536.1; SEQ ID NO: 24) and RABC (NCBI accession number: NP_002829.3; SEQ ID NO: 25) (Hermiston et al. 2003, “CD45: a critical regulator of signaling thresholds in im mune cells.” Annu Rev Immunol. 2: 107-137). CD45RA is expressed in naive T cells, and CD45RO is expressed in activated and memory T cells, some subsets of B cells, activated monocytes / macrophages, and granulocytes. CD45RB is expressed in peripheral B cells, naive T cells, thymocytes, and weakly in macrophages and dendritic cells. ) In one embodiment, provided herein is a binding region corresponding to that of Ab1, for example

[0191] ​​​​Namely, it is an anti-CD45 antibody or an antigen-binding fragment thereof that contains a CDR and a variable region. Ab The amino acid sequence of the heavy chain variable region (VH) of Ab1 is shown in SEQ ID NO: 7 (see Table 4). . The amino acid sequences of the VH CDR domains of Ab1 are shown in SEQ ID NO: 1 (CDR-H1), SEQ ID NO: 2 (CDR-H2), and SEQ ID NO: 3 (CDR-H3). The amino acid sequence of the light chain variable region (VL) of Ab1 is shown in SEQ ID NO: 8 (see Table 4). A b1's VL CDR domain amino acid sequences are SEQ ID NO: 4 (CDR-L1), SEQ ID NO: 5 (CDR-L2), and SEQ ID NO: 6 (CDR-L3). Thus, In one embodiment, the present disclosure is an anti-CD45 antibody or an antigen-binding fragment thereof used in combination with the compositions and methods described herein obtained, and includes an anti-CD45 antibody or an antigen-binding fragment thereof having one or more or all of the following CDRs: provided: a. CDR-H1 having the amino acid sequence FTFNNYWMT (SEQ ID NO: 1); b. CD R-H2 having the amino acid sequence SISSSGGSIYYPDSVKG (SEQ ID NO: 2); c. CDR-H3 having the amino acid sequence ARDERWAGAMDA (SEQ ID NO: 3); d. CDR-L1 having the amino acid sequence KASQNINKNLD (SEQ ID NO: 4); e. CDR-L2 having the amino acid sequence ETNNLQT (SEQ ID NO: 5); and f. CDR-L3 having the amino acid sequence YQHNSRFT (SEQ ID NO: 6).

[0192] In certain embodiments, the present disclosure is an anti-CD45 antibody or an antigen-binding fragment thereof that can be used in combination with the compositions and methods described herein and has the following heavy chain and A CD45 antibody or antigen thereof, including those having one or more or all of the light chain variable regions provides a binding fragment: Ab1 heavy chain (HC) variable region (CDRs underlined)

Chemical formula

Chemical formula

[0193] In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region containing an HC variable domain comprising SEQ ID NO: 7 or a variant of SEQ ID NO: 7, wherein the variant differs from SEQ ID NO: 7 in (i) 1, 2, 3, 4 or 5 amino acid substitutions, additions or deletions; ( ii) differs from SEQ ID NO: 7 in at most 5, 4, 3, 2 or 1 amino acid substitutions, additions or deletions; (iii) differs from SEQ ID NO: 7 in 1 - 5, 1 - 3, 1 - 2, 2 - 5 or 3 - 5 amino acid substitutions additions or deletions; and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 9 9% identical to SEQ ID NO: 7, wherein in any of (i)-(iv), the amino acid substitution can be a conservative amino acid substitution or a non - conservative amino acid substitution, and the modified heavy chain variable region has enhanced biological activity compared to that of SEQ ID NO: 7. In certain embodiments, the antibody comprises a modified light chain (LC) variable region containing an LC variable domain comprising SEQ ID NO: 8 or a variant of SEQ ID NO: 8, wherein the variant differs from SEQ ID NO: 8 in (i) 1, 2, 3, 4 or 5 amino acid substitutions, additions or deletions; ( ii) differs from SEQ ID NO: 8 in at most 5, 4, 3, 2 or 1 amino acid substitutions, additions or deletions; (iii) differs from SEQ ID NO: 8 in 1 - 5, 1 - 3, 1 - 2, 2 - 5 or 3 - 5 amino acid substitutions

[0194] additions or deletions; and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 9 9% identical to SEQ ID NO: 8, wherein in any of (i)-(iv), the amino acid substitution differs from SEQ ID NO: 8 in 2, 3, 4 or 5 amino acid substitutions, additions or deletions; ii) a sequence having up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions from SEQ ID NO: (iii) 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions; (iv) differs from SEQ ID NO: 8 in a sequence that is at least partially identical to SEQ ID NO: 8; At least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 9 9% identical amino acid sequence, wherein in any of (i) to (iv), The amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified light chain The variable region has enhanced biological activity compared to that of SEQ ID NO:8.

[0195] In certain embodiments, the anti-CD45 antibody comprises the CDRs described herein (SEQ ID NOS: 1-3 and and 4 to 6), and the CDRs contain conservative amino acid substitutions (or 2, 3, 4, or 5 (including amino acid substitutions).

[0196] In addition, the endonucleases on human CD45 that Ab1 (or antibodies having the Ab1 binding region) bind to Anti-human CD45 antibodies or fragments thereof that bind to a target antigen are also contemplated herein. Furthermore, anti-human C antibody that competes with Ab1 (or an antibody having the binding region of Ab1) is shown. The D45 antibody or an antigen-binding fragment thereof is contemplated.

[0197] In some embodiments, the anti-CD45 antibody or antigen-binding fragment thereof is The region containing the sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 27) of human CD45 For example, in certain embodiments, an anti-CD45 antibody or its antigen-binding The fragment specifically binds to human CD45 at amino acid residues 486R, 493Y, and 502T of SEQ ID NO: 26 (fragment of the CD45 isoform corresponding to NP_002829.3), or at corresponding residues in a region containing the like, of other human CD45 isoforms. In some embodiments, the anti-CD45 antibody or antigen-binding fragment thereof specifically binds to the fibronectin domain of human CD45 (e.g., the fibronectin d4 domain). Sequence of human CD45 isoforms:

Chemical formula

[0198] In one embodiment, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds to an epitope of human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 28) and binds to cynomolgus and rhesus CD45. In one embodiment, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds to an epitope of human CD45 comprising residues 486R, 493Y, and 502T of SEQ ID NO: 26

[0199] and also binds to cynomolgus and rhesus CD45. In one embodiment, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds to an epitope of human CD45 comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 27) and also binds to cynomolgus and rhesus CD45.

[0200] In one embodiment, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds to an epitope of human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 28) and binds to cynomolgus and rhesus CD45. In one embodiment, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds to an epitope of human CD45 comprising residues 486R, 493Y, and 502T of SEQ ID NO: 26 and binds to cynomolgus and rhesus CD45.

[0201] In one embodiment, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds to an epitope of human CD45 comprising residues 486R, 493Y, and 502T of SEQ ID NO: 26 Specifically binds to an episode of human CD45 containing residues 486R, 493Y, and 502T and binds to at least one additional amino acid, at least at least two additional amino acids, at least three additional amino acids, at least four additional amino acids, or at least five additional amino acids in a peptide containing RNGPHERYHLEVEAGNT (SEQ ID NO: 27) (the additional amino acid residues are not residues 486R, 493Y, and 502 T of SEQ ID NO: 26), and also binds to cynomolgus monkey and rhesus monkey CD45.

[0202] In some embodiments, the anti-CD45 antibody can bind to the extracellular domains of various isoforms of human CD45. Thus, in certain embodiments, the antibodies of the present specification are pan-specific anti-CD45 antibodies (i.e., antibodies that bind to all six human CD45 isoforms). Furthermore, Ab1 (or the binding region or specificity of this antibody antibody) can also bind to cynomolgus monkey CD45.

[0203] In an exemplary embodiment, the anti-CD45 antibody used in combination with the conditioning methods described herein is a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a fully human antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a fully human antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment a fully human antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment a bispecific antibody or its antigen-binding fragment, a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment , an F(ab’)2 molecule, or a tandem di-scFv. The A described herein DC or other exemplary anti-CD45 antibodies that can be used, in whole or in part, in the methods are provided below.

[0204] In one embodiment, the anti-CD45 antibody is clone HI30, available from BIOLEGEND (登録商標) (San Diego, California), or is derived therefrom, or a humanized variant thereof. Humanization of the antibody can be performed by substituting the framework residues and constant region residues of the non-human antibody with those of a germline human antibody according to procedures known in the art (e.g., as described in Example 7 below). Additional anti-CD45 antibodies that may be used in conjunction with the methods described herein include anti-CD45 antibodies ab10558, EP322Y, MEM-28, ab10559, 0.N. 125, F10-89-4, HIe-1, 2B11, YTH24.5, PD7 / 26 / 1 6, F10-89-4, 1B7, ab154885, B-A11, phosphor S 1007, ab170444, EP350, Y321, GA90, D3 / 9, X16 / 99, and LT45 (these are available from ABCAM (登録商標) (Cambridge, MA)), or humanized variants thereof, are included. Additional anti-CD45 antibodies that may be used in conjunction with the patient conditioning procedures described herein include anti-CD45 antibody HPA000440 (this is available from SIGMA-ALDRICH (登録商標) (St. Louis, MO)), and humanized variants thereof are included. ​​​​​​It may be used in conjunction with the patient conditioning method described herein. Further Examples of anti-CD45 antibodies include the mouse monoclonal antibody BC8 (which is described, for example, in Ma tthews et al., Blood 78:1864-1874, 1991, the disclosure of which is incorporated herein by reference as being relevant to anti-CD45 antibodies), and humanized variants thereof. Further anti-CD45 antibodies that may be used in conjunction with the methods described herein include the monoclonal antibody YAML 568 (which is described, for example, in Glatting et al., J. Nucl. Me d. 8:1335-1341, 2006, the disclosure of which is incorporated herein by reference as being relevant to anti-CD45 antibodies), and humanized variants thereof. Further anti-CD45 antibodies that may be used in conjunction with the patient conditioning procedures described herein include the monoclonal antibodies YTH54.12 and Y TH25.4 (which is described, for example, in Brenner et al., Ann. N.Y. Acad. Sci. 996:80-88, 2003, the disclosure of which is incorporated herein by reference as being relevant to anti-CD45 antibodies), and humanized variants thereof. Further anti-CD45 antibodies that may be used in conjunction with the patient conditioning method described herein include UCHL1, 2H4, SN130, MD4.3, MBI, and MT2 (which are described, for example, in Brown et al., Immunology 64:331-336, 1998, the disclosure of which is incorporated herein by reference as being relevant to anti-CD45 antibodies), and humanized variants thereof. Further anti-CD45 antibodies that may be used in conjunction with the patient conditioning method described herein include UCHL1, 2H4, SN130, MD4.3, MBI, and MT2 (which are described, for example, in Brown et al., Immunology 64:331-336, 1998, the disclosure of which is incorporated herein by reference as being relevant to anti-CD45 antibodies), and humanized variants thereof. including those that are incorporated and their humanized variants. In combination with the methods described herein Additional anti-CD45 antibodies that may be used include those produced and released from the American Type Culture Collection (ATCC) accession numbers RA3-6132, RA3-2C2, and TIB 122, as well as the anti-CD45 antibodies monoclonal antibodies C3 63.16A, and 13 / 2 (these are described, for example, in Johnson et al., J . Exp. Med. 169:1179-1184, 1989, and this disclosure is incorporated herein by reference as relating to anti-CD45 antibodies) , including those that are incorporated and their humanized variants. In combination with the patient conditioning methods described herein, additional anti-CD45 antibodies that may be used include monoclonal anti bodies AHN-12.1, AHN-12, AHN-12.2, AHN-12.3, AHN-1 2.4, HLe-1, and KC56 (T200) (these are described, for example, in Harvath et al., J. Immunol. 146:949-957, 1991, and this disclosure is incorporated herein by reference as relating to anti-CD45 antibodies), including those that are incorporated and their humanized variants. Additional anti-CD45 antibodies that may be used in combination with the patient conditioning methods described herein include, for example, U.S. Patent No. 7,265,212 (for example, anti-CD45 antibodies 39E11, 16C9, and 1G10 are described among other clones), U.S. Patent No.

[0205] CD45 antibodies include, for example, U.S. Patent No. 7,265,212 (for example, anti-CD45 antibodies 39E11, 16C9, and 1G10 are described among other clones), U.S. Patent No. where anti-CD45 antibodies 39E11, 16C9, and 1G10 are described among other clones), U.S. Patent U.S. Patent No. 7,160,987 (e.g., anti-CD45 antibodies such as monoclonal antibody 6G3, produced and released by ATCC accession number HB-11873), and U.S. Patent No. 6,099,838 (e.g., anti-CD45 antibody MT3, as well as antibodies produced and released by ATCC accession numbers HB220 (also designated as MB23G2) and HB2 23), and US2004 / 0096901 and US2008 / 0003224 (e.g., anti-CD45 antibodies produced and released by ATCC accession number PTA-7339, e.g., monoclonal antibody 17.1) are included, and the contents of each of these are incorporated herein by reference as being related to anti-CD45 antibodies.

[0206] Furthermore, anti-CD45 antibodies that can be used in combination with the patient conditioning methods described herein include antibodies produced and released from ATCC accession numbers MB4B4, MB23G2, 14.8 , GAP 8.3, 74-9-3, I / 24.D6, 9.4, 4B2, M1 / 9.3.4 .HL.2, and their humanized and / or affinity matured variants. Affinity maturation can be carried out, for example, using in vitro display technologies described herein such as phage display as described in Example 6 below or known in the art.

[0207] Additional anti-CD45 antibodies that can be used in combination with the patient conditioning methods described herein include anti-CD45 antibody T29 / 33, and the anti-CD45 antibody T29 / 33 is described, for example, in Morikawa et al., Int.J.Hematol. 5 :495 - 504, 1991, the disclosure of which is incorporated herein by reference as relating to anti - CD45 antibodies.

[0208] In certain embodiments, the anti - CD45 antibody is apamistamab (also known as 90Y - BC8, Iomab - B, BC8; for example, US20170326259, WO2017155937, and Orozco et al. Blood. 127 .3 (2016):352 - 359) or BC8 - B10 (for example, Li et al. PloS one 13.10 (2018):e0205135), each of which is incorporated by reference. Other anti - CD45 anti bodies are described, for example, in WO2003 / 048327, WO2016 / 016442, US201 7 / 0226209, US2016 / 0152733, US9,701,756, US2 011 / 0076270, or US7,825,222, each of which is incorporated herein by reference in its entirety.

[0209] For example, in one embodiment, it is an anti - CD45 antibody or an antigen - binding fragment thereof that contains a binding region corresponding to that of apamistamab, such as a CDR , variable region. The amino acid sequence of the heavy - chain variable region (VH) of apamistamab is shown in SEQ ID NO: 31 (see Table 4 ). The amino acid sequence of the light - chain variable region (VL) of apamistamab is described in SEQ ID NO: 32 (see Table 4). In other embodiments, the anti - CD45 antibody or its antigen - binding portion is ​​​​A variable heavy chain containing the amino acid residue shown in SEQ ID NO: 31 and a light chain variable region. In one embodiment, the anti-CD45 antibody comprises a heavy chain comprising CDR1, CDR 2 and CDR3 of apamistamab, and a light chain variable region comprising CDR1, CDR2 and CDR3 of apamistamab.

[0210] In one embodiment, the anti-CD45 antibody comprises the heavy chain of the anti-CD45 antibody described herein and the light chain variable region of the anti-CD45 antibody described herein. In one embodiment, the anti-CD45 antibody comprises a heavy chain comprising CDR1, CDR2 and CDR3 of the anti-CD45 antibody described herein and a light chain variable region comprising CDR1, CDR2 and CDR3 of the anti-CD45 antibody described herein.

[0211] In another embodiment, the antibody or antigen-binding fragment thereof has at least 95% identity to the anti-CD45 antibody herein, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to the anti-CD45 antibody herein, and comprises a heavy chain variable region having an amino acid sequence. In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region containing the HC variable domain (HC) of the anti-CD45 antibody or a variant thereof herein, the variant differing from the anti-CD45 antibody in (i) 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) at least about 75%, 80 % identity to the anti-CD45 antibody; and / or (iv) at least about 75%, 80 % identity to the anti-CD45 antibody; and / or (iv) at least about 75%, 80 % identity to the anti-CD45 antibody; and / or (iv) at least about 75%, 80 % identity to the anti-CD45 antibody; and / or (iv) at least about 75%, 80 % identity to the anti-CD45 antibody; and / or (iv) at least about 75%, 80 % identity to the anti-CD45 antibody; and / or (iv) at least about 75%, 80 % identity to the anti-CD45 antibody; and / or (iv) at least about 75%, 80 ​​​amino acids that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical comprising a sequence, wherein in any of (i)-(iv), the amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution, and the modified heavy chain variable region can have enhanced biological activity compared to the heavy chain variable region of an anti-CD45 antibody while retaining the CD45 binding specificity of the antibody.

[0212] In one embodiment, the methods and compositions disclosed herein specifically bind to human CD45 (and possibly CD45 from one or more non-human species), but do not substantially bind to non-CD45 proteins, and comprise an anti-CD45 antibody or an antigen-binding fragment thereof. In embodiments, the antibody or fragment thereof has a KD of 1×10−6 M or less, 5×10−7 M or less, 3×10−7 M or less, 1×10−7 M or less, 5×10−8 M or less, 1×10−8 M or less, or 1×10−9 M or less for binding to human CD45. -7 D D - 8 D D -8 D D -8 D D -9 D D -10 D D -11 D D for binding to human CD45.

[0213] Furthermore, in certain embodiments, the anti-CD45 ADC has a serum half-life in a human subject of about 3 days or less. In certain embodiments, the anti-CD45 described herein has a serum half-life of about 24 hours or less, about 23 hours or less, about 22 hours or less, about 21 hours or less, about 20 hours or less, about 19 hours or less. ​​​​​​​​​​​​​Less than about 18 hours, less than about 17 hours, less than about 16 hours, less than about 15 hours, less than about 14 hours or having a half-life of less than about 13 hours, less than about 12 hours, or less than about 11 hours (e.g., in humans).

[0214] In one embodiment, the anti-CD45 antibodies described herein have a half-life of about 1-5 hours, about 5-10 hours, about 10-15 hours, about 15-20 hours, or about 20-25 hours (e.g., in humans).

[0215] Additional anti-CD45 antibodies that can be used in the ADCs described herein can be identified using techniques known in the art, including hybridoma production. Hybridomas can be prepared using mouse strains. Protocols for immunization and subsequent isolation of spleen cells for fusion are known in the art. Fusion partners and procedures for hybridoma generation are also known. Alternatively, anti-CD45 antibodies can be generated using HuMAb-Mouse® or XenoMouse® (商標) In the production of additional anti-CD45 antibodies, the CD45 antigen is isolated and / or purified. The CD45 antigen can be a fragment of CD45 from the extracellular domain of CD45. Immunization of animals can be performed by any method known in the art. For example, see Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as mice, rats, sheep, goats,[[ID=XREF=37]] pigs, cows, horses, etc. are well known in the art. For example, the above-mentioned Harlow and Lane reference can be used.​​​​​ See also Lane and U.S. Patent No. 5,994,619. The CD45 antigen can be administered with an adjuvant to stimulate an immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl di peptide), or ISCOM (immunostimulatory complex). After immunizing an animal with the CD45 antigen, an antibody-producing immortalized cell line is prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized by methods known in the art (e.g., introduction of oncogenes, introduction of oncogenic viruses, exposure to carcinogenic or mutagenic compounds, fusion with immortalized cells (e.g., myeloma cells), inactivation of tumor suppressor genes). See, e.g., Harlow and Lane supra. Hybridomas can be selected, cloned, and further screened for desired properties such as robust growth, high antibody production, and desired antibody characteristics. The anti-CD45 antibodies for use in the anti-CD45 ADCs described herein can also be identified using high-throughput screening of libraries of antibodies or antibody fragments that can bind to a molecule that binds to CD45. Such methods include in vitro display deep technologies known in the art such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, cDNA display, etc. The use of phage display to isolate antibodies, antigen-binding fragments, or ligands that bind to a molecule related to biology is described, for example, in Felici et al., Biote

[0216] The anti-CD45 antibodies for use in the anti-CD45 ADCs described herein can also be identified using high-throughput screening of libraries of antibodies or antibody fragments that can bind to a molecule that binds to CD45. Such methods include in vitro display deep technologies known in the art such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, cDNA display, etc. The use of phage display to isolate antibodies, antigen-binding fragments, or ligands that bind to a molecule related to biology is described, for example, in Felici et al., Biote phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, cDNA display, etc., in vitro display deep technologies known in the art. Antibodies, antigen-binding fragments, or ligands that bind to molecules related to biology can be isolated using phage display. For example, see Felici et al., Biote include in vitro display deep technologies known in the art such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, cDNA display, etc. To isolate an antibody, antigen-binding fragment, or ligand that binds to a molecule related to biology, the use of phage display is described, for example, in Felici et al., Biote niques 82: 301-310 (1991) and Ladner et al., U.S. Patent No. 5,223,409. Chnol. Annu. Rev. 1:149 - 183, 1995; Katz , Annu. Rev. Biophys. Biomol. Struct. 2 6:27 - 45, 1997; and Hoogenboom et al., Imm unotechnology 4:1 - 20, 1998, which are each incorporated herein by reference as related to in vitro display technology. Kay, Perspect. Drug Discover y Des. 2:251 - 268, 1995 and Kay et al., Mol l. Divers. 1:139 - 140, 1996 (each of these disclosures is incorporated herein by reference as related to the discovery of antigen - binding molecules). As described in Kay, Perspect. Drug Discovery Des. 2:251 - 268, 1995 and Kay et al., Mol l. Divers. 1:139 - 140, 1996 (each of these disclosures is incorporated herein by reference as related to the discovery of antigen - binding molecules), to select polypeptides that bind to cell - surface antigens, a randomized combinatorial peptide library has been constructed. Proteins such as multimeric proteins have been successfully used in phage display as functional molecules (e.g., EP0349 578; EP4527839; and EP0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80 - 84, 1992; each of these disclosures is incorporated herein by reference as related to the use of in vitro display technology for the discovery of antigen - binding molecules). Furthermore, functional antibody fragments such as Fab and scFv are expressed in in vitro display formats (e.g., McCafferty et al., Trends Biotechnol. 10:80 - 84, 1992; each of these disclosures is incorporated herein by reference as related to the use of in vitro display technology for the discovery of antigen - binding molecules). . Furthermore, functional antibody fragments such as Fab and scFv are expressed in in vitro display formats (e.g., McCafferty et al., ​Nature 348:552-554, 1990; Barbas et al. , Proc. Natl. Acad. Sci. USA 88:7978-798 2, 1991; and Clackson et al., Nature 352: 624-628, 1991 (see). Each of these disclosures is incorporated herein by reference as related to in vitro display platforms for the discovery of antigen-binding molecules. incorporated herein.

[0217] In addition to in vitro display technologies, computational modeling technologies can be used to design and identify, for example, anti-CD 45 antibodies or fragments using the procedures described in US2013 / 0288373, and the disclosure of US2013 / 0288 373 is incorporated herein as related to molecular modeling methods for identifying anti-CD45 antibodies. For example, using computational modeling technologies, one of ordinary skill in the art can screen a library of antibodies or antibody fragments in silico for molecules that can bind to specific epitopes on CD45 (e.g., extracellular epitopes of CD45). can be screened.

[0218] In one embodiment, the anti-CD45 antibody used in the ADCs described herein can be internalized into cells. In the identification of anti-CD45 antibodies (or fragments thereof), additional techniques can be used to identify antibodies or antigen-binding fragments that bind to CD45 on the surface of cells (e.g., lymphocytes) and can be internalized by the cells, for example, via receptor-mediated endocytosis. For example, the in vitro dis ​​​​​​​Spray technology can be adapted to screen antibodies or their antigen-binding fragments that bind to CD45 on the surface of hematopoietic stem cells and are then internalized. Phage display is one such technique that can be used in combination with this screening paradigm. To identify antibodies or their fragments that bind to CD45 and are then internalized into CD45+ cells, one of ordinary skill in the art can use the phage display technology described in Williams et al ., Leukemia 19:1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety.

[0219] The internalization ability of the prepared antibodies or their fragments can be evaluated, for example, using radionuclide uptake assays known in the art. For example, anti-CD45 antibodies or their fragments identified using in vitro display techniques described herein or known in the art can be labeled with F, 18 F, 75 Br, 7 7 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 17 7 Lu, 77 As, 72 As, 86 Y,90 Y, 89 Zr, 212 Bi, 213 Bi, or or 225 can be functionalized by incorporation of radioisotopes such as Ac. For example, 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 radioactive halogens such as At can be incorporated into an antibody, or a fragment thereof, using beads such as polystyrene beads containing an electrophilic halogen reagent (e.g., iodinated beads, Thermo Fisher Scientific, Cambridge, Massachusetts). The radiolabeled antibody or fragment thereof can be incubated with T cells and / or NK cells for a time sufficient to allow internalization. The internalized antibody or fragment thereof can be identified by detecting the radiation emitted from the resulting hematopoietic stem cells (e.g., γ-rays) and comparing it to the radiation emitted from the recovered wash buffer (e.g., γ-rays). In some embodiments, the anti-CD45 antibody (or fragment thereof) has a defined serum half-life. For example, the anti-CD45 antibody (or fragment thereof) can have a serum half-life of about 1 to 24 hours in a human patient. An ADC containing such an anti-CD45 antibody can also, for example, have a serum half-life of about 1 to 24 hours in a human patient. Pharmacokinetic analysis by measurement of serum levels can be performed by assays known in the art. For example, γ-rays) and comparing it to the radiation emitted from the recovered wash buffer (e.g., γ-rays).

[0220] In some embodiments, the anti-CD45 antibody (or fragment thereof) has a defined serum half-life. For example, the anti-CD45 antibody (or fragment thereof) can have a serum half-life of about 1 to 24 hours in a human patient. An ADC containing such an anti-CD45 antibody can also, for example, have a serum half-life of about 1 to 24 hours in a human patient. Pharmacokinetic analysis by measurement of serum levels can be performed by assays known in the art. .​​​​​

[0221] For recombinant production of anti-CD45 antibodies, nucleic acids encoding the antibodies, e.g., as described above, can be used alone. isolated and stored in one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be inserted into the target using conventional procedures (e.g., antibody heavy chain and By using an oligonucleotide probe capable of specifically binding to the gene encoding the light chain, and (by) can be easily isolated and sequenced.

[0222] Suitable host cells for cloning or expressing antibody-encoding vectors include those described herein. For example, antibodies may be prepared from prokaryotic or eukaryotic cells, particularly those described in the literature, including those described in the literature, such as those described in the literature, particularly those described in the literature, such as ... If Fc effector functions are not required, antibodies can be produced in bacteria. For fragment and polypeptide expression, see, e.g., U.S. Pat. No. 5,648,232. See U.S. Patent No. 7, U.S. Patent No. 5,789,199, and U.S. Patent No. 5,840,523. See Charlton, which describes the expression of antibody fragments in E. coli. , Methods in Molecular Biology, Vol. 248 (BK C. Lo, ed., Humana Press, Totowa, NJ, 2003 (See also pp. 245-254.) After expression, the antibody is isolated from the bacterial cell paste in a soluble fraction. It can be further purified.

[0223] Vertebrate cells can also be used as hosts, e.g., cells grown in suspension. Adapted mammalian cell lines may be useful. Other examples of useful mammalian host cell lines include S. monkey kidney CV1 strain transformed by V40 (COS-7); human embryonic kidney strain (e.g. then, 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (197 7); baby hamster kidney cells (BHK) ; mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1) ; African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W13 8); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); e.g., , TRI cells as described in Mather et al., Annals N.Y. Acad. Sci. 3 83:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR-CHO cells Chinese hamster ovary (CHO) cells (Urlaub et al., Proc . Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y 0, NS0, Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Meth ods in Molecular Biology, Vol. 248 (B.K.C. Lo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In one embodiment, the host cell is a eukaryote, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (Y0, NS0, Sp20 cells, etc.).

[0224] ​Fc engineered antibodies

[0225] The present disclosure provides an Fc polypeptide capable of binding to an antigen expressed by lymphocytes, such as CD45. The antibody or antigen-binding fragment thereof having Fc modifications that allow for immune binding is i) to treat cancer and autoimmune diseases, and (ii) for patients requiring transplantation therapy as a therapeutic agent alone or as an ADC to promote engraftment of transplanted hematopoietic stem cells in These therapeutic activities are based in part on the discovery that they can be used as therapeutic agents for, e.g., cell ( an anti-CD45 antibody or its anti-CD45 antibody that binds to CD45 expressed by lymphocytes It can be caused by the binding of the original binding fragments.

[0226] The antibodies or binding fragments described herein also include antibodies and / or fragments. Modifications and / or mutations that change the properties of the compound, for example, increasing half-life or AD It may contain modifications and / or mutations that increase or decrease CC.

[0227] In one embodiment, the anti-CD45 antibody or binding fragment thereof comprises a modified Fc region: The modified Fc region comprises at least one amino acid modification compared to a wild-type Fc region, As a result, the molecule has altered affinity or binding to Fc gamma R (FcγR). Certain amino acid positions within the Fc region have been shown to make direct contact with FcγRs through crystallography studies. Specifically, amino acids 234-239 (hinge region), amino acid 265 -269 (B / C loop), amino acids 297-299 (C' / E loop), and amino The amino acid 327-332 (F / G) loop (Sondermann et al., 20 (See Nature, 406:267-273 in 00). In some embodiments, the antibodies described herein may include a variant Fc region containing a modification of at least one residue that directly contacts FcγR . In one embodiment , the Fc region of the anti-CD45 antibody (or fragment thereof) includes an amino acid substitution at amino acid 265 according to the EU index as in Kabat et al., Sequences of Prote ins of Immunological Interest, 5th Edition, Public Health Service, USA, NH1, Maryland (1991), which is incorporated herein by reference expressly. “EU index as in Kabat” refers to the numbering of human IgG1 EU antibodies. In one embodiment, the Fc region includes the D26 5A mutation. In one embodiment, the Fc region includes the D265C mutation. In some embodiments , the Fc region of the antibody (or fragment thereof) includes an amino acid substitution at amino acid 234 according to the EU index as in Kabat. In one embodiment, the Fc region includes the D265C mutation. In some embodiments , the Fc region of the antibody (or fragment thereof) includes an amino acid substitution at amino acid 234 according to the EU index as in Kabat.

[0228] In one embodiment, the Fc region includes mutations at the amino acid positions of D265, V205, H435, I253, and / or H310. For example, specific mutations at these positions include D265C, V205C, H435A, I253A, and / or H310A .

[0229] In one embodiment, the Fc region includes the L234A mutation. In some embodiments, the Fc region of the anti-CD 45 antibody (or fragment thereof) includes an amino acid substitution at amino acid 235 according to the EU index as in Kabat. In one embodiment, the Fc The region contains the L235A mutation. In yet another embodiment, the Fc region comprises L234A and the L235A mutation. In a further embodiment, the Fc region comprises D265C, L234A and the L235A mutation. In yet another embodiment, the Fc region comprises D265C, L23 4A, L235A and the H435A mutation. In a further embodiment, the Fc region comprises D 265C and the H435A mutation.

[0230] In yet another embodiment, the Fc region comprises the L234A and L235A mutations (also referred to herein as “L234A.L235A” or “LA”). In another embodiment , the Fc region comprises the L234A and L235A mutations, where the Fc region does not comprise the P329 G mutation. In a further embodiment, the Fc region comprises D265C, L234A and the L235A mutation (also referred to herein as “D265C.L234A.L235A”) . In another embodiment, the Fc region comprises D265C, L234A and L235A mutations , where the Fc region does not comprise the P329G mutation. In yet another embodiment, the F c region comprises D265C, L234A, L235A and the H435A mutation (also referred to herein as “D265C.L234A.L235A.H435A”). In another embodiment the Fc region comprises D265C, L234A, L235A and the H435A mutation, where the Fc region does not comprise the P329G mutation. In a further embodiment, the Fc region is , D265C and the H435A mutation (also referred to herein as “D265C.H435A”) is included. In yet another embodiment, the Fc region comprises D265A, S239C, L23 It includes the 4A and L235A mutations (also referred to herein as "D265A.S239C.L234A.L2 35A"). In yet another embodiment, the Fc region includes the D265A, S2 39C, L234A, and L235A mutations, where the Fc region does not include the P329G mutation. In another embodiment, the Fc region includes the D265C, N297G, and H435 A mutations (also referred to herein as "D265C.N297G.H435A"). In another embodiment, the Fc region includes the D265C, N297Q, and H435A mutations (also referred to herein as "D265C.N297Q.H435A"). In another embodiment the Fc region includes the E233P, L234V, L235A, and delG236 (deletion of 236 mutation (also referred to herein as "E233P.L234V.L235A.delG236" or "EPLVLAdelG"). In another embodiment, the Fc region includes the E233P, L234V, L235A, and delG236 (deletion of 236) mutations and where the Fc region does not include the P329G mutation. In another embodiment, the Fc region includes the E233P, L234V, L235A, delG236 (deletion of 236), and H435 A mutations (also referred to herein as "E233P.L234V.L235A.delG236.H43 5A" or "EPLVLAdelG.H435A"). In another embodiment the Fc region includes the E233P, L234V, L235A, delG236 (deletion of 236), and H435A mutations, where the Fc region does not include the P329G mutation . In another embodiment, the Fc region includes the L234A, L235A, S239C, and D265 A mutations. In another embodiment, the Fc region includes the L234A, L235A, S239C, and comprising the Yob and D265A mutations, wherein the Fc region does not comprise the P329G mutation. In another embodiment, the Fc region comprises the H435A, L234A, L235A and D265C mutations In another embodiment, the Fc region comprises the H435A, L234A, L235A and D2 65C mutations, wherein the Fc region does not comprise the P329G mutation.

[0231] In some embodiments, the antibody has a modified Fc region that reduces effector function by reducing binding to the Fc receptor (FcR) of the same antibody comprising a non-modified Fc region, as compared to binding to the FcR in an in vitro effector function assay. In some embodiments, the antibody has a modified Fc region that reduces effector function by reducing binding to the Fc gamma receptor (FcγR) of the same antibody comprising a non-modified Fc region, as compared to binding to the FcγR in an in vitro effector function assay. In some embodiments, the FcγR is FcγR1. In some embodiments, the FcγR is FcγR2A. In some embodiments, the FcγR is compared to binding to the FcγR of the same antibody comprising a non-modified Fc region in an in vitro effector function assay. In some embodiments, the FcγR is FcγR2B. In other embodiments, the FcγR is FcγR2C. In some embodiments, the FcγR is FcγR3A. In some embodiments, the FcγR is Fc γR3B. In other embodiments, the reduction in binding is at least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least 98 % reduction, at least 99% reduction, or 100% reduction in antibody binding to the FcγR as compared to binding to the FcγR of the same antibody comprising a non-modified Fc region. In other embodiments, the reduction in binding is at least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least 98 % reduction, at least 99% reduction, or 100% reduction in antibody binding to the FcγR as compared to binding to the FcγR of the same antibody comprising a non-modified Fc region. % reduction, at least 99% reduction, or 100% reduction in antibody binding to the FcγR as compared to binding to the FcγR of the same antibody comprising a non-modified Fc region. In other embodiments, The decrease in binding is at least 70% to 100% less, at least 80% to 100% less, at least 90% to 100% less, at least 95% to 100% less, or at least 98% to 100% less in the binding of the antibody to FcγR compared to the binding of the same antibody containing an unmodified Fc region to Fcγ R. In some embodiments, the antibody has a modified Fc region that decreases cytokine release by at least 50% in an in vitro cytokine release assay compared to the cytokine release of the same antibody containing an unmodified Fc region. In some embodiments the decrease in cytokine release is at least 70% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, at least 99% less, or 100% less in the cytokine release of the antibody compared to the cytokine release of the same antibody containing an unmodified Fc region. In some embodiments, the decrease in cytokine release is at least 70% to 100% less, at least 80% to 100% less, at least

[0232] 90% to 100% less, at least 95% to 100% less in the cytokine release of the antibody compared to the cytokine release of the same antibody containing an unmodified Fc region. In certain embodiments the cytokine release is by immune cells. In some embodiments, the antibody has a modified Fc region that decreases mast cell degranulation by at least 50% in an in vitro mast cell degranulation assay compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments the antibody has a modified Fc region that decreases mast cell degranulation by at least 50% in an in vitro mast cell degranulation assay compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments the decrease in mast cell degranulation is at least 70% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, at least 99% less, or 100% less in the mast cell degranulation of the antibody compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments, the decrease in mast cell degranulation is at least 70% to 100% less, at least 80% to 100% less, at least 90% to 100% less, at least 95% to 100% less in the mast cell degranulation of the antibody compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In certain embodiments the mast cell degranulation is by immune cells. In some embodiments, the antibody has a modified Fc region that decreases mast cell degranulation by at least 50% in an in vitro mast cell degranulation assay compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments the decrease in mast cell degranulation is at least 70% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, at least 99% less, or 100% less in the mast cell degranulation of the antibody compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments, the decrease in mast cell degranulation is at least 70% to 100% less, at least 80% to 100% less, at least 90% to 100% less, at least 95% to 100% less in the mast cell degranulation of the antibody compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In certain embodiments the mast cell degranulation is by immune cells. In some embodiments, the antibody has a modified Fc region that decreases mast cell degranulation by at least 50% in an in vitro mast cell degranulation assay compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments

[0233] the antibody has a modified Fc region that decreases mast cell degranulation by at least 50% in an in vitro mast cell degranulation assay compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments the decrease in mast cell degranulation is at least 70% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, at least 99% less, or 100% less in the mast cell degranulation of the antibody compared to the mast cell degranulation of the same antibody containing an unmodified Fc region. In some embodiments The reduction in mast cell degranulation is compared to mast cell degranulation of the same antibody containing an unmodified Fc region and is at least a 70% reduction, at least an 80% reduction, at least a 90 % reduction, at least a 95% reduction, at least a 98% reduction, at least a 99% reduction , or a 100% reduction. In some embodiments, the reduction in mast cell degranulation is at least 70% to 100%, at least 80% to 100%, at least 90% to 1 00%, or at least 95% to 100% reduction in mast cell degranulation compared to mast cell degranulation of the same antibody containing an unmodified Fc region.

[0234] In some embodiments, the antibody has a modified Fc region that reduces or prevents antibody-dependent cell phagocytosis (ADCP) by at least 50% in an in vitro antibody-dependent cell phagocytosis assay compared to the antibody-dependent cell phagocytosis (ADCP) of the same antibody containing an unmodified Fc region. In some embodiments, the reduction in ADCP is at least a 70% reduction, at least an 80 % reduction, at least a 90% reduction, at least a 95% reduction, at least a 98% reduction , at least a 99% reduction, or a 100% reduction in cytokine release compared to cytokine release of the same antibody containing an unmodified Fc region.

[0235] In some embodiments, the anti-HC antibody (e.g., anti-CD45 antibody) described herein comprises an Fc region containing one of the following modifications or combinations of modifications: D265A , D265C, D265C / H435A, D265C / LALA, D265C / LALA / H435A, D265A / S239C / L234A / L235A / H435A, D26 ​​​​​5A / S239C / L234A / L235A, D265C / N297G, D265C / N 297G / H435A, D265C(EPLVLAdelG*), D265C(EPLV LAdelG) / H435A, D265C / N297Q / H435A, D265C / N2 97Q, EPLVLAdelG / H435A, EPLVLAdelG / D265C, EP LVLAdelG / D265A, N297A, N297G, or N297Q. In some embodiments, the anti-CD45 antibodies herein have an Fc region containing one of the following modifications or combinations of modifications: D265A, D265C, D265C / H435A D265C / LALA, D265C / LALA / H435A, D265C / N297G D265C / N297G / H435A, D265C(IgG2 ), D265C(Ig * )、D265C(Ig G2) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPLVLAdelG / H435A, N297A, N297G, or N297Q.

[0236] The binding or affinity between the modified Fc region and the Fc gamma receptor can be determined by various techniques known in the art (e.g., without limitation, equilibrium methods (e.g., enzyme-linked immuno absorbent assay (ELISA)); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2 008; radioimmunoassay (RIA)), or surface plasmon resonance assays or other kinetic-based assays of mechanisms ​​​A (e.g., BIACORE (商標) analysis or Octet (商標) analysis (forteB IO)), and other methods (e.g., indirect binding assay, competitive binding assay, fluorescence resonance energy transfer (fluorescence resonance energy tr ansfer (FRET)), gel electrophoresis and chromatography (e.g., gel filtration )) can be used for measurement. These and other methods may utilize labels on one or more components of the test subject, and / or various detection methods (e.g., chromogenic labels , fluorescent labels, luminescence labels, or isotope labels, etc., including but not limited to these) may be utilized. Detailed descriptions regarding binding affinity and kinetics can be found in Paul, W . E., ed., Fundamental Immunology, 4th E d., Lippincott-Raven, Philadelphia (1999 )(which focuses on antibody-immunogen interactions). An example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen with the antibody of interest while increasing the abundance of unlabeled antigen , and detecting the antibody bound to the labeled antigen. The affinity and binding off-rate of the antibody of interest for a specific antigen may be determined from data by Scatchard plot analysis . Competition with a second antibody may also be measured using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound while increasing the abundance of unlabeled second antibody .

[0237] ​​​​​ In one embodiment, an antibody having an Fc modification described herein (e.g., D265C, L234A, L2 35A, and / or H435A) has a binding to the Fc gamma receptor that is (e.g., when evaluated by biolayer interferometry (BLI)) at least 70%, at least 80 %, at least 90%, at least 95%, at least 98%, at least 99%, or also 100% reduced compared to the binding of the same antibody with an unmodified Fc region to the Fc gamma receptor.

[0238] Without wishing to be bound by any theory, the Fc region binding interaction with the Fc gamma receptor is thought to be essential for various effector functions and downstream signal transduction events including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, in certain embodiments, an antibody comprising a modified Fc region (e.g., comprising the L234A, L235A, and / or D265C mutations ) has substantially reduced or abolished effector function. Effector function can be assayed using various methods known in the art, for example, by measuring a cellular response to the antibody of interest (e.g., mast cell degranulation or cytokine release). For example, using standard methods in the art, an Fc-modified antibody can be assayed for its ability to cause mast cell degranulation or, for example, cytokine release by human peripheral blood mononuclear cells.

[0239] Thus, in one embodiment, the Fc region is (e.g., compared to an antibody having an unmodified include mutations that result in a decrease in half-life compared to). Antibodies with short half-lives are expected to function as therapeutic agents in certain cases where the antibody is short-lived and are advantageous in the conditioning steps described herein where CAR therapy is performed following administration of the antibody It can become. Ideally, the antibody is substantially removed prior to the administration of CAR therapy, and the antibody generally also expresses the target antigen (e.g., CD45), but unlike endogenous stem cells, it is not the target of the anti-CD45 antibody. In one embodiment, the Fc region contains a mutation at position 435 (EU index according to Kabat) . In one embodiment, this mutation is the H435A mutation.

[0240] In one embodiment, the anti-CD45 described herein has a half-life (e.g., in humans) of about 24 hours or less, about 23 hours or less, about 22 hours or less, about 21 hours or less, about 20 hours or less, about 19 hours or less, about 18 hours or less, about 17 hours or less, about 16 hours or less, about 15 hours or less, about 14 hours or less, about 13 hours or less, about 12 hours or less or about 11 hours or less.

[0241] In one embodiment, the anti-CD45 antibody described herein has a half-life (e.g., in humans) of about 1-5 hours, about 5-10 hours , about 10-15 hours, about 15-20 hours, or about 20-25 hours. In one embodiment, the half-life of the anti-HC antibody is about 5-7 hours, about 5-9 hours, about 5-11 hours, about 5-13 hours, about 5-15 hours, about 5-20 hours, about 5-24 hours, about 7-24 hours, about 9-24 hours, about 11-24 hours, about 12-22 hours, about 10 -20 hours, about 8-18 hours, or about 14-24 hours.

[0242] In some embodiments, the Fc region has a shortened half-life and a reduced effector function of the antibody comprises two or more mutations that result in. In some embodiments, the Fc region has a decreased half-life a mutation that results in, and a mutation of at least one residue that can directly contact FcγR (e.g., based on structural and crystallographic analysis ). In one embodiment, the Fc region comprises the H435A mutation , the L234A mutation, and the L235A mutation. In one embodiment, the Fc region comprises H4 35A mutation and the D-265C mutation. In one embodiment, the Fc region comprises the H435A mutation, L 234A mutation, L235A mutation, and D265C mutation.

[0243] In some embodiments, the antibody or its antigen-binding fragment is conjugated to a cytotoxin (e.g., amatoxin) via a cysteine residue in the Fc domain of the antibody or its antigen-binding fragment . In some embodiments, the cysteine residue is introduced by a mutation in the Fc domain of the antibody or its antigen-binding fragment. For example , the cysteine residue can be selected from the group consisting of Cys118, Cys239, and Cys265 . In one embodiment, the Fc region of an anti-CD45 antibody (or its fragment) has an amino acid substitution at amino acid 265 according to the EU index as in Kabat . In one embodiment, the Fc region comprises the D265C mutation. In one embodiment, the Fc region comprises the D265C and H435A mutations. In one embodiment, the Fc region comprises D26 5C, L234A, and L235A mutations. In one embodiment, the Fc region comprises D265 C, L234A, L235A, and H435A mutations. In one embodiment, the Fc region of an anti-CD45 antibody or its antigen-binding fragment has an EU index as in Kabat antibody or its antigen-binding fragment has an EU index as in Kabat ​​In accordance with the Dex, it includes amino acid substitutions at amino acid 239. In one embodiment, the Fc region contains the S239C mutation. In one embodiment, the Fc region contains the L234A mutation, L235A mutation mutation, S239C mutation and D265A mutation. In another embodiment, the Fc region contains S2 39C and H435A mutations. In another embodiment, the Fc region contains the L234A mutation, L235A mutation and S239C mutation. In yet another embodiment, the Fc region contains H 435A mutation, L234A mutation, L235A mutation and S239C mutation. In yet another embodiment, the Fc region contains H435A mutation, L234A mutation, L235A mutation, S2 39C mutation and D265A mutation.

[0244] In particular, the positions of the Fc amino acids refer to the EU numbering index unless otherwise specified.

[0245] Antibodies and antigen-binding fragments that can be used in combination with the compositions and methods described herein include the antibodies and antigen-binding fragments thereof described above, as well as humanized variants of the non-human antibodies and antigen-binding fragments described above, and antibodies or antigen-binding fragments that bind to the same epitope as those described above, as evaluated by, for example, competitive antigen-binding assays. fragments are included.

[0246] The antibodies of the present disclosure are described, for example, in (Dall’Acqua et al. (2006) J Biol Chem 281:23514-24), (Zalevsky et al .(2010) Nat Biotechnol 28:157-9), (Hinto n et al. (2004) J Biol Chem 279:6213-6), (Hinton et al. (2006) J Immunol 176:346 - 5 6), (Shields et al. (2001) J Biol Chem 27 6:6591 - 604), (Petkova et al. (2006) Int I mmunol 18:1759 - 69), (Datta - Mannan et al. (2007) Drug Metab Dispos 35:86 - 94), (Vac caro et al. (2005) Nat Biotechnol 23:1283 - 8), (Yeung et al. (2010) Cancer Res 70:3 269 - 77) and (Kim et al. (1999) Eur J Immun ol 29:2819 - 25), such as those described in, additional Fc mutations can be introduced to further manipulate and further regulate the antibody half - life, positions 250, 25 2, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435 are included. Exemplary mutations that can be made alone or in combination are T250Q , M252Y, 1253A, S254T, T256E, P2571, T307A, D37 6V, E380A, M428L, H433K, N434S, N434A, N434H, N 434F, H435A and H435R mutations.

[0247] Methods of engineering an antibody to include any of the Fc modifications herein are well known in the art. These methods include, but are not limited to, site - specific (or oligonucleo tide) modification of the antibody or at least the DNA molecule encoding the constant region of the anti Preparation by mutagenesis via deoxynucleotides, PCR mutagenesis, and cassette mutagenesis is included . Site-directed mutagenesis is known in the art (see, for example, Carter et al . , Nucleic Acids Res., 13:4431-4443 (1985 year) and Kunkel et al., Proc. Natl. Acad. Sci. U SA, 82:488 (1987)). PCR mutagenesis is also suitable for generating amino acid sequence variants of the starting polypeptide. See Higuchi, PCR Protocols, 177-183 (Academic Press, 1990), and Vall ette et al., Nuc. Acids Res. 17:723-733 ( 1989). Cassette mutagenesis, another method for preparing sequence variants, is based on the technique described by Wells et al., Gene, 34:315-323 (198 5).

[0248] Cytotoxin

[0249] Various cytotoxins can be conjugated to the anti-CD45 antibody via a linker for use in the combination therapies described herein. In particular, an anti-CD45 ADC comprises an antibody (or antigen-binding fragment thereof) conjugated (i.e., covalently bound via a linker) to a cytotoxic moiety (or cytotoxin). In various embodiments, the cytotoxic moiety has reduced or no cytotoxicity when conjugated, but recovers cytotoxicity after cleavage from the linker. In various embodiments, the cytotoxic moiety maintains cytotoxicity without cleavage from the linker. In some embodiments ​​​​​​​​​, The cytotoxic molecule is conjugated to the intracellular antibody or antigen-binding fragment thereof disclosed herein, such that subsequent to the uptake of the antibody or its fragment into the cell, the cytotoxin can access its intracellular target and mediate, for example, T cell death. , The antibodies, antigen-binding fragments and ligands thereof (e.g., antibodies that recognize and bind CD45, antigen-binding fragments and soluble ligands thereof) described herein can be conjugated (or linked) to cytotoxins. , The antibodies, antigen-binding fragments and ligands thereof (e.g., antibodies that recognize and bind CD45, antigen-binding fragments and soluble ligands thereof) described herein can be conjugated (or linked) to cytotoxins.

[0250] Thus, the ADCs of the present disclosure can be of the general formula Ab-(Z-L-D) , where the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently) to a linker (L) via a chemical moiety (Z) and conjugated to a cytotoxic moiety (the "drug", D). "n" represents the number of drugs linked to the antibody and generally ranges from 1 to 8. , where the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently) to a linker (L) via a chemical moiety (Z) and conjugated to a cytotoxic moiety (the "drug", D). "n" represents the number of drugs linked to the antibody and generally ranges from 1 to 8.

[0251] Thus, the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from n , where the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently) to a linker (L) via a chemical moiety (Z) and conjugated to a cytotoxic moiety (the "drug", D). "n" represents the number of drugs linked to the antibody and generally ranges from 1 to 8. , where the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently) to a linker (L) via a chemical moiety (Z) and conjugated to a cytotoxic moiety (the "drug", D). "n" represents the number of drugs linked to the antibody and generally ranges from 1 to 8. , where the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently) to a linker (L) via a chemical moiety (Z) and conjugated to a cytotoxic moiety (the "drug", D). "n" represents the number of drugs linked to the antibody and generally ranges from 1 to 8. , where "n" represents the number of drugs linked to the antibody and generally ranges from 1 to 8.

[0252] , where the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from , where the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from , where the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from , where the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from , where the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from , where the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from , where the antibody or antigen-binding fragment thereof can be conjugated to a number of drug moieties indicated by the integer n, where n represents the average number of cytotoxins per antibody and can range, for example, from about 1 to about 20. In some embodiments, n is from 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some examples, homogeneous ADCs with other drug loads having a particular value of n can be obtained from Separation, purification, and characterization of potential ADCs can be performed by methods such as reversed-phase HPLC or electrophoresis. This can be achieved by:

[0253] For some anti-CD45 ADCs, the average number of cytotoxins per antibody is For example, if the linkage is a cysteine thiol, the antibody may have only one or several cysteine thiol groups or through which Only one or a few sufficiently reactive thiols to which linkers and chemical moieties can be attached Generally, antibodies have many free reactive cytosines that can be linked to drug moieties. primarily, cysteine thiol residues in antibodies are involved in disulfide bridges In certain embodiments, the antibody is present as a dithiocarbamate under partial or full reducing conditions. Reduced phosphates such as diisopropyl phosphate (DTT) or tricarbonylethylphosphine (TCEP) It can be reduced with a base to generate a reactive cysteine thiol group. In the case of high drug loading (e.g., n>5), aggregation of certain antibody-drug conjugates occurs. This may result in insolubility, toxicity, or loss of cell permeability.

[0254] In certain embodiments, fewer drug moieties than the theoretical maximum are present in the conjugation reaction. During the reaction, the antibody is conjugated to an antibody, for example, a drug linker, as described below. The most reactive lysine group may contain lysine residues that do not react with the intermediate or linker reagent. can react with an amine-reactive linker reagent. or subjected to denaturing conditions to reveal reactive nucleophilic groups such as cysteine. It will be.

[0255] The ADC payload (drug / antibody ratio) can be controlled by different methods, for example, (i) limiting the molar excess of the drug-linker intermediate or linker reagent to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) using partial or restrictive reducing conditions for cysteine thiol modification, (iv) manipulating the amino acid sequence of the antibody by recombinant techniques such that the number and / or position of cysteine residues is altered for control of the number and / or position of linker-drug linkages.

[0256] Cytotoxins suitable for use in the compositions and methods described herein include, among others, DNA intercalating agents (e.g., anthracyclines), agents that can disrupt the mitotic spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin, and their derivatives), and agents that can interfere with protein biosynthesis (e.g., agents that exhibit rRNA N-glycosidase activity such as saporin and ricin A-chain), which are known in the art.

[0257] In some embodiments, the cytotoxins are microtubule binding agents (e.g., maytansine or maytansinoids), amatoxins, Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, auristatin, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, indolinobenzodiazepine ​​​​​​​​​​​​​​​Zepin dimer, indolinobenzodiazepine pseudodimer, or a variant thereof, or another cytotoxic compound described in the present specification or known in the art. iazepine pseudodimer), or another cytotoxic compound described in the present specification or known in the art. is a cytotoxic compound described in the present specification or known in the art.

[0258] Additional details regarding the cytotoxins that can be used in anti-CD45 ADCs useful in the methods of the present disclosure are described below. are described below.

[0259] Amatoxin

[0260] In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor. is an RNA polymerase inhibitor.

[0261] In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody-drug conjugate disclosed herein is amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody-drug conjugate disclosed herein is amatoxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, proamanullin, or a derivative thereof. The structures of various naturally occurring amatoxins are represented by Formula II and the accompanying Table 1, for example, Zanotti et al ., Int. J. Peptide Protein Res. 30, 1987 ., pages 450-459. ., Int. J. Peptide Protein Res. 30, 1987 , pages 450-459.

Chemical Formula

[0262] Table 1. Table of amatoxin structures

Table 1

[0263] Amatoxins are found in various mushroom species (e.g., Amanita phalloides). phalloides), Galerina marginata, It can also be isolated from Lepiota brunneo-incarnata and semi-synthetically Alternatively, it can be prepared synthetically. A member of this family, α-amanitin, Wieland, Int. J. Pept. Protein Res.198 3, 22(3):257-276. Amatoxin derivatives are naturally occurring obtained by chemical modification of compounds present in nature ("semi-synthetic"), or from entirely synthetic sources Synthetic routes to various amatoxin derivatives are described, for example, in U.S. Pat. No. 9,676,702 and Perrin et al., J.Am.Chem.S oc. 2018, 140, pp. 6513-6517, each of which is No. 6,239,999, incorporated herein by reference in its entirety for synthetic methods for preparing and derivatizing amatoxins. incorporated herein.

[0264] Many positions on an amatoxin or derivative thereof can be linked to a linking moiety L and thus to an antibody or its derivative. These can serve as sites for covalently attaching antigen-binding fragments of the target molecule. In embodiments, the cytotoxin in the ADCs disclosed herein is an amphotericin represented by formula (III): Toxins or their derivatives: [ka] During the ceremony: R1 is H, OH, or OR A is; R2 is H, OH, or OR B ; R A and R B , when present, together with the oxygen to which they are attached, optionally form a substituted 5-membered heterocycloalkyl group; R3 is H or R D ; R4 is H, OH, OR D , or R D ; R5 is H, OH, OR D , or R D ; R6 is H, OH, OR D , or R D ; R7 is H, OH, OR D , or R D ; R8 is OH, NH2, or OR D ; R9 is H, OH, or OR D ; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkyn yl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), any optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0265] For example, in one embodiment, an a useful in combination with the compositions and methods described herein Matoxin includes compounds according to formula (IIIA):

Chemical formula

[0266] For example, in one embodiment, the alpha useful in combination with the compositions and methods described herein Matoxin includes compounds according to the following formula (IIIB):

Chemical formula

[0267] In one embodiment, amatoxins useful in combination with the compositions and methods described herein also include compounds of the following formula (IIIC): : [Chemical formula] Wherein: R1 is H, OH, or OR A ; R2 is H, OH, or OR B ; R A and R B , when present, together with the oxygen to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D ; R4 is H, OH, OR D , or R D ; R5 is H, OH, OR D , or R D ; R6 is H, OH, OR D , or R D ; R7 is H, OH, OR Dor R D is; R8 is OH, NH2, or OR D is; R9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO2-; and R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkyn yl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), any optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0268] In one embodiment, the cytotoxin is amanitin.

[0269] As described herein, amatoxin can be conjugated to an antigen-binding fragment, an antibody or an antigen-binding fragment thereof, for example via a linker moiety. Exemplary methods of amatoxin conjugation and linkers useful in such processes are described in the section entitled "Linkers for Chemical Conjugation" and in Table 1 below. Exemplary linker-containing amatoxins useful for conjugation to an anti-CD45 antibody or antigen-binding fragment according to the compositions and methods described herein are the structural formulas (I), (IA), (IB), (IV), (IVA) described herein Exemplary methods of amatoxin conjugation and linkers useful in such processes are described in the section entitled "Linkers for Chemical Conjugation" and in Table 1 below. Exemplary linker-containing amatoxins useful for conjugation to an anti-CD45 antibody or antigen-binding fragment according to the compositions and methods described herein are the structural formulas (I), (IA), (IB), (IV), (IVA) described herein are described in the section entitled "Linkers for Chemical Conjugation" and in Table 1 below. Exemplary linker-containing amatoxins useful for conjugation to an anti-CD45 antibody or antigen-binding fragment according to the compositions and methods described herein are the structural formulas (I), (IA), (IB), (IV), (IVA) described herein are described in the section entitled "Linkers for Chemical Conjugation" and in Table 1 below. Exemplary linker-containing amatoxins useful for conjugation to an anti-CD45 antibody or antigen-binding fragment according to the compositions and methods described herein are the structural formulas (I), (IA), (IB), (IV), (IVA) described herein or antigen-binding fragment are the structural formulas (I), (IA), (IB), (IV), (IVA) described herein described herein and are shown in (IVB).

[0270] For example, an antibody or antigen-binding fragment described herein can be conjugated to amatoxin to form a conjugate represented by the formula Ab-Z-L-Am where Ab is an antibody or an antigen-binding fragment thereof, L is a linker, Z is a chemical moiety and Am is amatoxin. Many positions on amatoxin or its derivatives can function as sites for covalently linking the linking moiety L and thus the antibody or an antigen-binding fragment thereof. In some embodiments, the amatoxin-linker conjugate Am-L-Z is represented by formula (I)

Chemical formula

[0271] In some embodiments, the cytotoxin comprises one R C substituent.

[0272] In some embodiments, R A and R B together with the oxygen atom to which they are attached form the following:

Chemical formula

[0273] In some embodiments, Am-L-Z is represented by formula (I), wherein: R1 is H, OH, OR A , or OR C ; R2 is H, OH, OR B , or OR C ; R A and R B together with the oxygen atom to which they are attached form

Chemical formula

[0274] In some embodiments, Am-LZ is represented by formula (I): During the ceremony: R1 is H, OH, OR A , or OR C is; R2 is H, OH, OR B , or OR C is; R A and R B together with the oxygen atoms to which they are attached, [ka] form; R3 is H or R C is; R4 and R5 each independently represent H, OH, OR C , R C , or OR D is ; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and X and R C is as defined above.

[0275] In some embodiments, Am-LZ is represented by formula (I): During the ceremony: R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; RA and R B together with the oxygen atoms to which they are attached form

Chemical formula

[0276] In some embodiments, Am-L-Z is represented by formula (I), wherein: R1 and R2 are each independently H or OH; R3 is R C ; R4, R6 and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; and R C is as defined above.

[0277] In some embodiments, Am-L-Z is represented by formula (I), wherein: R1 and R2 are each independently H or OH; R3, R6 and R7 are each H; R4 and R5 are each independently H, OH, OR C , or R C ; R8 is OH or NH2; R9 is H or OH; and R C is as defined above. Such amatoxin conjugates include For example, see U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is , the entirety of which is incorporated herein by reference.

[0278] In some embodiments, Am-LZ is represented by formula (I): During the ceremony: R1 and R2 are each independently H or OH; R3, R6 and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and R C is as defined above. Such amatoxin conjugates include For example, as described in U.S. Pat. No. 9,233,173 and U.S. Pat. No. 9,399,681 No. 6,239,999, the disclosure of each of which is incorporated herein by reference in its entirety.

[0279] In some embodiments, the linker L and chemical moiety Z together as LZ are [ka] wherein S is a nucleotide present in an antibody or antigen-binding fragment thereof that binds to CD45. The sulfur atom represents a reactive substituent (e.g., in the -SH group of a cysteine residue).

[0280] In some embodiments, LZ is [Chemical formula] is.

[0281] In some embodiments, L-Z is [Chemical formula] is.

[0282] In some embodiments, Am-L-Z is represented by formula (IA), [Chemical formula] wherein: R1 is H, OH, OR A , or OR C is; R2 is H, OH, OR B , or OR C is; R A and R B , when present, together with the oxygen to which they are attached, optionally form a substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , R C , or R D is; R6 is H, OH, OR C , OR D , R C , or R D is; R7 is H, OH, OR C , OR D , R C , or RD is; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D is; R9 is H, OH, OR C , or OR D is; X is -S-, -S(O)-, or -SO2-; R C is -L-Z; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkyn yl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalk ylene), optionally substituted cycloalkylene, optionally substituted heterocycloalk ylene, optionally substituted arylene, optionally substituted heteroarylene, peptide (e.g For example, dipeptides, -(C=O)-, disulfides, hydrazones, -(CH2CH2O ) p - group [wherein p is an integer from 1 to 6], ((CH2) m O) n (CH2) m -group [n and and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. or combinations thereof; and Z is a reactive substituent Z' present on L and an antibody or antigen binding site thereof that binds to CD45. Chemical groups formed from coupling reactions between reactive substituents present in the coupling fragments is part; Am contains exactly one Rc substituent.

[0283] In some embodiments, Am-LZ is represented by formula (IB): [ka] During the ceremony: R1 is H, OH, OR A , or OR C is; R2 is H, OH, OR B , or OR C is; R A and R B When present, together with the oxygen atoms to which they are attached, forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4 is H, OH, OR C , OR D , R C , or R D is; R5 is H, OH, OR C , OR D , RC or R D ; R6 is H, OH, OR C OR D R C or R D ; R7 is H, OH, OR C OR D R C or R D ; R8 is OH, NH2, OR C OR D NHR C or NR C R D ; R9 is H, OH, OR C or OR D ; X is -S-, -S(O)-, or -SO2-; R C is -L-Z; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkyn yl), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), any optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., (C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalk ynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalk ylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -(C=O)-, peptide, disulfide, hydrazone, -(CH2CH2O) p - group p is an integer from 1 to 6], ((CH2) m O) n (CH2) m - group [n and each m are, each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10] or a linker such as a combination thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent Z' present on L and a reactive substituent present within an antibody or antigen-binding fragment thereof that binds to CD45 ; Am contains exactly one Rc substituent.

[0284] In some embodiments, the linker contains -(CH) 2n - units, where n is an integer from 2 to 6. In some embodiments, the linker contains -((CH2) n and where , n is 6.

[0285] In some embodiments, the linker L and the chemical moiety Z are together as L-Z,

Chemical formula

[0286] In some embodiments, L-Z is

Chemical formula

[0287] In some embodiments, L-Z is

Chemical formula

[0288] In some embodiments, the conjugate Am-L-Z-Ab is represented by any one of the following structural formulas as follows:

Chemical formula

[0289] In some embodiments, Am-L-Z-Ab is represented by the following structural formula:

Chemical formula

[0290] In some embodiments, Am-L-Z', which is a precursor of Am-L-Z-Ab, is

Chemical formula

[0291] In some embodiments, Am-L-Z is represented by formula (IV), formula (IVA), or formula (IV VB),

Chemical formula

[0292] In some embodiments, the linker contains -(CH2)- units, where n is an integer from 2 to 6. In some embodiments, R1 is the linker, R2 is H, and the linker and chemical moiety together are L-Z as n [Chem.]

[0293] In some embodiments, R1 is the linker, R2 is H, and the linker and chemical moiety together are L-Z as [Chem.]

[0294] In some embodiments, Am-L-Z-Ab is [Chemical formula] as follows.

[0295] In some embodiments, Ab-Z-L-Am is [Chemical formula] as follows.

[0296] In some embodiments, Am-L-Z-Ab is [Chemical formula] as follows.

[0297] In some embodiments, the Am-L-Z-Ab precursor (i.e., Am-L-Z’) is one of the following: [Chemical formula] Here, the maleimide reacts with the thiol group present in the cysteine of the CD45 antibody.

[0298] According to the compositions and methods described herein, additional amatoxins that can be used for conjugation to an antibody or an antigen-binding fragment thereof are, for example, WO201 6 / 142049, WO2016 / 071856, and WO2017 / 046658, the disclosures of each of which are incorporated herein by reference in their entirety. For example, antibodies, antigen-binding fragments thereof, and ligands that recognize and bind CD45 are α-amanitin or a variant thereof, as described in US2015 / 0218220 Can be conjugated to a rhenium, the disclosure of US2015 / 0218220, for example, α - amatoxins such as amatoxins and their variants, such as α - amanitin, and covalent conjugates Related to covalent linkers that can be used in the tion, are incorporated herein by reference The method for synthesizing amatoxin is described, for example, in US Patent No. 9,676,702, which patent is incorporated herein by reference with respect to the synthesis methods disclosed herein Included.

[0299] The linker L can be attached to an amatoxin (e.g., an amatoxin of formula III, IIIA, IIIB, or IIIC) at any one of several possible positions (e.g., any of R 1 ~R 9 ), to provide an amatoxin - linker conjugate of formula I, IA, IB, IV, IVA, or IVB.

[0300] In some embodiments, the linker is attached at position R 1 . In some embodiments , the linker is attached at position R 2 . In some embodiments, the linker is attached at position R 3 . In some embodiments, the linker is attached at position R 4 . In some embodiments, the linker is attached at position R 5 . In some embodiments, the lin ker is attached at position R 6 . In some embodiments, the linker is attached at position R 7 . In some embodiments, the linker is attached at position R 8 . In some embodiments , the linker is attached at position R 9 .

[0301] In some embodiments, the cytotoxin is α - amanitin. In some embodiments , the linker comprises hydrazine, disulfide, thioether or dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val - Ala and Val - Cit . In some embodiments, the linker comprises a para - aminobenzyl group (PAB ). In some embodiments, the linker comprises the moiety PAB - Cit - Val . In some embodiments, the linker comprises the moiety PAB - Ala - Val. In some embodiments, the linker comprises -((C=O)(CH2) n - units, where n is an integer from 1 to 6.

[0302] In some embodiments, the linker comprises -(CH2) n - units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB - Cit - Val - ((C=O)(CH2) n -. In some embodiments, the linker is -PAB - Ala - Val -((C=O)(CH2) n -. In some embodiments, the linker -L and the chemical moiety Z together form L - Z as [Chemical formula] .

[0303] Auristatin

[0304] The anti - CD45 antibodies and antigen - binding fragments described herein are auristatins can be conjugated to a cytotoxin (U.S. Patent No. 5,635,483; U.S. Patent No. 5 ,780,588). Auristatin interferes with microtubule dynamics, GTP hydrolysis, as well as nuclear and cell division (Woyke et al (2001) Antimi crob. Agents and Chemother. 45(12):3580 - 3 584), has anticancer activity (U.S. Patent No. 5,663,149) and antifungal activity (Pett it et al (1998) Antimicrob. Agents Chemot her. 42:2961 - 2965), and is an antimitotic agent (U.S. Patent No. 5,6 35,483; U.S. Patent No. 5,780,588). The auristatin drug moiety can be attached to an antibody via the N(amino) - terminus or C(carboxyl) - terminus of the peptide drug moiety (WO02 / 088172).

[0305] Exemplary embodiments of auristatin include the N - terminal - linked monomethyl auristatin drug moieties DE and DF (MMAE and MMAF, respectively) disclosed in Senter et al, Proceedings of the American As sociation for Cancer Research, Volume 45 , Abstract Number 623, published on March 28, 2004, the disclosure of which is hereby incorporated by reference in its entirety.

[0306] Exemplary embodiments of auristatin are MMAE:

Chemical formula

[0307] Another exemplary auristatin embodiment is MMAF: [Chemical formula] and wherein the "wavy line" indicates the point of covalent attachment to the linker of an antibody linker conjugate (-L-Z-Ab as disclosed in US2005 / 0238649 and described herein ).

[0308] Auristatins can be prepared according to the methods of U.S. Patent No. 5,635,483; U.S. Patent No. 5,780,588 ; Pettit et al (1989) J. Am. Chem. Soc. 1 11:5463-5465; Pettit et al (1998) Anti-Ca ncer Drug Design 13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pet tit et al (1996) J. Chem. Soc. Perkin Tra ns. 15:859-863; and Doronina (2003) Nat. B iotechnol. 21(7):778-784.

[0309] Maytansinoid

[0310] The antibodies and antigen-binding fragments thereof described herein can be conjugated to cytotoxins that are microtubule-binding agents. In some embodiments, the microtubule-binding agent is maytansine, It is a maytansinoid or a maytansinoid analog. A maytansinoid is a mitotic inhibitor that acts by binding to microtubules and inhibiting tubulin polymerization. Maytansine was first isolated from the African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was also discovered that certain microorganisms produce maytansinoids such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Patent No. 4,137,230; U.S. Patent No. 4 ,248,870; U.S. Patent No. 4,256,746; U.S. Patent No. 4,260,608 ; U.S. Patent No. 4,265,814; U.S. Patent No. 4,294,757; U.S. Patent No. 4 ,307,016; U.S. Patent No. 4,308,268; U.S. Patent No. 4,308,269 ; U.S. Patent No. 4,309,428; U.S. Patent No. 4,313,946; U.S. Patent No. 4 ,315,929; U.S. Patent No. 4,317,821; U.S. Patent No. 4,322,348 ; U.S. Patent No. 4,331,598; U.S. Patent No. 4,361,650; U.S. Patent No. 4 ,364,866; U.S. Patent No. 4,424,219; U.S. Patent No. 4,450,254 ; U.S. Patent No. 4,362,663; and U.S. Patent No. 4,371,533. The maytansinoid drug moiety is relatively easy to prepare by (i) fermentation or chemical modification and derivatization of fermentation products, (ii) is easily derivatized with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) is stable in plasma, and (iv) is effective against various tumor cell lines . , (iv) is effective against various tumor cell lines, and (v) is suitable for conjugation to antibodies via non-disulfide linkers . ​​​​​​ is an attractive drug moiety of an antibody-drug conjugate.

[0311] Examples of suitable maytansinoids include esters of maytansinol, synthetic maytansinoids, as well as maytansinol analogs and derivatives. As used herein, any cytotoxin that inhibits microtubule formation and has high toxicity to mammalian cells, such as maytansinoids, maytansinol, and maytansinol analogs and derivatives, is included. .

[0312] Examples of suitable maytansinol esters include those having a modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are described in U.S. Patent No. 4,137,230; U.S. Patent No. 4,151,042; U.S. Patent No. 4,248,87 0; U.S. Patent No. 4,256,746; U.S. Patent No. 4,260,608; U.S. Patent No. 4,265,814; U.S. Patent No. 4,294,757; U.S. Patent No. 4,307,01 6; U.S. Patent No. 4,308,268; U.S. Patent No. 4,308,269; U.S. Patent No. 4,309,428; U.S. Patent No. 4,313,946; U.S. Patent No. 4,315,92 9; U.S. Patent No. 4,317,821; U.S. Patent No. 4,322,348; U.S. Patent No. 4,331,598; U.S. Patent No. 4,361,650; U.S. Patent No. 4,362,66 3; U.S. Patent No. 4,364,866; U.S. Patent No. 4,424,219; U.S. Patent No. 4,450,254; U.S. Patent No. 4,322,348; U.S. Patent No. 4,362,66 3; U.S. Patent No. 4,371,533; U.S. Patent No. 5,208,020; U.S. Patent No. 5,416,064; U.S. Patent No. 5,475,092; U.S. Patent No. 5,585,49 No. 9; U.S. Patent No. 5,846,545; U.S. Patent No. 6,333,410; U.S. Patent No. 7,276,497; and U.S. Patent No. 7,473,796, each of which disclosures is hereby incorporated by reference as related to maytansinoids and their derivatives. In some embodiments, the antibody-drug conjugate (ADC) of the present disclosure utilizes, as the cytotoxic agent, a thiol-containing maytansinoid formally called N '-deacetyl...

Claims

1. A method for promoting the acceptance of immune cells expressing a chimeric antigen receptor (CAR) in a human subject having cancer or an autoimmune disease, the method comprising: (a) administering to the human subject having cancer or an autoimmune disease a therapeutically effective amount of an anti-CD45 antibody-drug conjugate (ADC), wherein the anti-CD45 ADC comprises an anti-CD45 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and

2. The method of claim 1, wherein the human subject is not administered alemtuzumab before, at the same time as, or after step (b).

3. The method of claim 1 or 2, wherein the human subject is not administered a lymphodepleting chemotherapeutic agent before, at the same time as, or after step (b).

4. The method of claim 3, wherein the lymphodepleting chemotherapeutic agent is fludarabine, cyclophosphamide, bendamustine, and / or pentostatin.

5. The method according to any one of claims 1 to 4, further comprising administering the anti-CD45 ADC to the human subject before step (b).

6. The method according to any one of claims 1 to 5, comprising administering the anti-CD45 ADC to the human subject about 12 hours to about 21 days before step (b).

7. The method according to any one of claims 1 to 6, wherein the immune cells are allogeneic cells or autologous cells.

8. The method of claim 7, wherein the allogeneic cells are allogeneic T cells or allogeneic NK cells.

9.

10. A method of treating a human patient having a tumor, the method comprising: (i) administering to the human patient a therapeutically effective amount of an anti-CD45 ADC, wherein the anti-CD45 ADC comprises an anti-CD45 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and (ii) administering to the human patient a therapeutically effective amount of immune cells expressing a CAR, wherein the CAR comprises an extracellular domain that binds to a tumor antigen or an antigen associated with an autoimmune disease, a transmembrane domain, and a cytoplasmic domain.

11. The method of claim 10, wherein the immune cells are allogeneic cells or autologous cells.

12. The anti-CD45 ADC is administered to the patient as a single dose or as multiple doses ​ ​ ​ ​ ​ ​ ​ ​ ​ The therapeutically effective amount of the allogeneic cells expressing the CAR is about 1×10 ４ to about 7.0×1 0 ８ The method according to any one of claims 1 to 8, which is cells / kg. ​ ​ ​ ​ comprising; and (ii) from about 1×10 ６ to about 7×10 ８ CAR T cells / kg ​ ​ The therapeutically effective amount of the CAR T cells is about 1×10 ６ to about 1×10 ８ cells / kg. ​ ​ ​ The method according to any one of claims 1 to 11.

13. The method according to any one of claims 1 to 12, wherein the human patient does not develop neutropenia after administration of the immune cells expressing the CAR.

14. The method according to any one of claims 1 to 12, wherein the human patient does not develop severe neutropenia after administration of the immune cells expressing the CAR.

15. Neutropenia is defined as a human patient having an absolute neutrophil count (ANC) of less than about 1500 (about 1500 / microliter) per microliter, the method according to claim 13.

16. Severe neutropenia is defined as an ANC of less than 500 / microliter, the method according to claim 15.

17. A method of lymphodepleting a human patient selected for CAR-T therapy, comprising administering a therapeutically effective amount of an anti-CD45 ADC to the human patient prior to administration of CAR-T cells to the human patient.

18. The method according to claim 17, wherein the human patient is not administered cyclophosphamide and / or fludarabine as a lymphodepletion regimen as a pretreatment for the CAR-T therapy.

19. The method according to claim 17, wherein the human patient is not subjected to lymphocyte depletion chemotherapy as a lymphodepletion regimen as a pretreatment for the CAR-T therapy.

20. The method according to any one of claims 17 to 19, further comprising administering CAR-T therapy to the human patient.

21. The anti-CD45 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3, respectively, and a light chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively, the method according to any one of claims 1 to 20.

22. The method according to claim 21, wherein the anti-CD45 antibody or antigen-binding fragment thereof is chimeric or humanized.

23. The anti-CD45 antibody or antigen-binding fragment thereof is of the IgG1 isotype or IgG4 isotype, the method according to any one of claims 1 to 22.

24. The cytotoxin is an anti-mitotic agent, a ribosome-inactivating protein (RIP), or R ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The method according to any one of claims 1 to 23, which is an RNA polymerase inhibitor.

25. The method according to claim 24, wherein the RNA polymerase inhibitor is amatoxin.

26. The method according to claim 24, wherein the RNA polymerase inhibitor is amanitin.

27. The amanitin according to claim 26, which is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amanine amide, amanullin, amanullinic acid, proamanullin, and derivatives thereof.

28. The method according to any one of claims 1 to 25, wherein the anti-CD45 ADC is represented by formula (I). 【Chemical 1】 (In the formula: R １ is H, OH, OR Ａ or OR Ｃ ; R ２ is H, OH, OR Ｂ or OR Ｃ ; R Ａ and R Ｂ When present, together with the oxygen atoms to which they are attached, any forms an optionally substituted 5-membered heterocycloalkyl group; R ３ is H, R Ｃ or R Ｄ ; R ４ 、R ５ 、R ６ 、and R ７ are each independently H, OH, OR Ｃ 、OR Ｄ 、R Ｃ or R Ｄ is; R ８ is OH, NH ２ , OR Ｃ , OR Ｄ , NHR Ｃ or NR Ｃ R Ｄ wherein; R ９ is H, OH, OR Ｃ or OR Ｄ ; X is -S-, -S(O)-, or -SO ２ -; R Ｃ is -L-Z; R Ｄ is optionally substituted alkyl (e.g., C １ -C ６ alkyl), optionally substituted heteroalkyl (e.g., C １ -C ６ heteroalkyl), optionally substituted alkenyl (e.g., C ２ -C ６ alkenyl), optionally substituted heteroalkenyl (e.g., C ２ -C ６ heteroalkenyl), optionally substituted alkynyl (e.g., C ２ -C ６ alkyn (l), optionally substituted heteroalkynyl (e.g., C ２ -C ６ heteroalkynyl), any is an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; L is optionally substituted alkylene (e.g., C １ -C ６ alkylene), optionally substituted the resulting heteroalkylene (C １ -C ６ heteroalkylene), optionally substituted alkenylene (e.g., C ２ -C ６ alkenylene), optionally substituted heteroalkenylene (e.g., C ２ -C ６ heteroalkenylene), optionally substituted alkynylene (e.g., C ２ -C ６ alkynylene), optionally substituted heteroalkynylene (e.g., C ２ -C ６ heteroal quinylene), an optionally substituted cycloalkylene, an optionally substituted heterocycloalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10] and a linker selected from combinations thereof; and -(C=O)-, peptide, disulfide, hydrazone, -(CH ２ CH ２ O) ｐ - group p is an integer from 1 to 6], ((CH ２ )) ｍ O) ｎ (CH ２ )) ｍ -group [n and each m are, Z is a chemical moiety formed from a coupling reaction between a reactive substituent Z' present on L and a reactive substituent present within the anti-CD45 antibody or an antigen-binding fragment thereof. )

29. The method according to claim 26, wherein the anti-mitotic agent is maytansine or auristatin.

30. The method according to claim 29, wherein the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).

31. The method according to claim 26, wherein the anti-mitotic agent is pyrrolobenzodiazepine (PBD) or calicheamicin.

32. The method according to any one of claims 1 to 31, wherein the linker is N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB) together with the reactive substituent Z' of the ADC.

33. The method according to any one of claims 1 to 32, wherein the ADC has a serum half-life of 3 days or less.

34. The extracellular domain of the CAR comprises a scFv antibody or a single-chain T cell receptor (scT CR), the method according to any one of claims 1 to 33. **Claim 35** The extracellular domain comprises a non-immunoglobulin scaffold protein, claim 1 - The method according to any one of 33. **Claim 36** The tumor antigen is CD19, CD22, CD30, CD7, BCMA, CD137, C D22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EG FR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, An antigen selected from the group consisting of TAA, NKG2D, or CD123, claim 1 - The method according to any one of 33. **Claim 37** The cytoplasmic domain of the CAR comprises a CD28 cytoplasmic signaling domain, CD3 zeta cytoplasmic signaling domain, OX40 cytoplasmic signaling domain, and / or a CD137 (4-1BB) cytoplasmic signaling domain, any one of claims 1 to 36 - The method described in the item. **Claim 38** The cytoplasmic domain of the CAR comprises a CD3 zeta cytoplasmic signaling domain - The method according to any one of claims 1 to 37. **Claim 39** The human subject having cancer is leukemia, advanced adult cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphoid cancer, liver metastasis of colorectal cancer ​ ​ ​ ​ ​ ​ ​ ​ Lymphoma, colorectal cancer, gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, lung Squamous cell carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic Leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell pre-lymphocytic Leukemia, pediatric acute lymphoblastic leukemia, refractory pediatric acute lymphoblastic leukemia, acute leukemia Acute lymphoblastic leukemia, acute lymphocytic leukemia, pre-lymphocytic leukemia, chronic lymphocytic ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The method according to claim 44, comprising an agent.

48. The method according to claim 44, wherein the T cell depletion therapy comprises an anti-CD4 antibody.

49. The method according to claim 44, wherein the T cell depletion therapy comprises an anti-CD8 antibody.

50. The method according to claim 44, wherein the T cell depletion therapy comprises an anti-CD137 antibody.

51. The method according to claim 44, wherein the T cell depletion therapy comprises an anti-CD52 antibody.

52. The method according to claim 51, wherein the anti-CD52 antibody is alemtuzumab.

53. The method according to claim 44, wherein the T cell depletion therapy comprises anti-thymocyte globulin (ATG). method.

54. The method according to claim 53, wherein the ATG is rabbit ATG (rATG).

55. The method according to claim 53, wherein the ATG is equine ATG (eATG).

56. The method according to claim 44, wherein the T cell depletion therapy comprises total body irradiation (TBI).

57. The level of one or more CAR-T engraftment cytokines in the human subject increases after administration of the anti-CD 45 ADC, according to any one of claims 1 to 56.

58. The CAR-T engraftment cytokine is IL-15 or IL-7, according to claim 57. described method.

59. The levels of one or more cytokine release syndrome (CRS)-cytokines do not substantially increase in the human patient after administration of the anti-CD 45 ADC, according to any one of claims 1 to 58. described method.

60. The one or more CRS-cytokines are IFNγ, IL-10, IL-6, IL-8 , MIP-1α, MIP-1β, or IL-10, according to claim 59.

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